method to detect a target in a sample and kit

专利摘要:
This description refers to compositions that enhance the deposition of detectable chemical portions in tissue samples, with the methods using these compositions and kits that include these compositions. The compositions include a deposition enhancer that has a formula and / or in which R ^ 1 ^, R ^ 2 ^, R ^ 3 ^ and R ^ 4 ^ are independently selected from a chemical portion containing heteroatom, aliphatic, aryl, halogen and hydrogen; R ^ 1 ^ and / or R ^ 3 ^ can be attached to R ^ 2 ^ to form a fused aromatic ring system; R ^ 5 ^ is selected from a chemical portion that contains heteroatom; A is selected from a carbon atom, a heteroatom, except sulfur, and any combination thereof; n is 1 to 5, an enzyme, a specific binding chemical portion and a detectable chemical portion.
公开号:BR112013014360B1
申请号:R112013014360-6
申请日:2011-12-28
公开日:2020-10-13
发明作者:Eric J. May；Adrian E. Murillo；Jerome W. Kosmeder
申请人:Ventana Medical Systems, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDER
[001] This application claims the benefit of the previous filing date of Provisional Application n5U.S. 61 / 460,349, which was filed on December 30, 2010. All disclosure of the provisional application is considered part of the disclosure of the following application and is incorporated by reference in this document. FIELD OF THE INVENTION
[002] This disclosure relates to innovative compositions containing pyrimidine analogs for use in increasing the deposition of detectable portions on target molecules in a tissue. BACKGROUND OF THE INVENTION
[003] Cell staining methods that include immunohistochemistry (IHC) and in situ hybridization analysis (ISH), are useful tools in histological diagnosis and in the study of tissue morphology. IHC employs specific binding agents or moieties, such as antibodies, to detect an antigen of interest that may be present in a tissue sample. IHC is widely used in diagnostic and clinical applications, such as for diagnosing particular conditions or disease states. For example, particular types of cancer can be diagnosed based on the presence of a particular marker molecule in a sample taken from an individual. IHC is also widely used in basic research to understand the location and distribution of a biological marker in different tissues. Biological samples can also be examined using sets of in situ hybridization procedures, such as silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH) and fluorescent in situ hybridization (FISH), collectively referred to as ISH . ISH is different from IHC, in that ISH detects nucleic acids in tissue while IHC detects proteins.
[004] For in situ assays such as HCI assays and ISH assays for tissue and cytological samples, especially multiplexed assays for such samples, it is highly desirable to identify and develop methods that provide desirable results without background interference. Such a method involves the use of Tiram Outgoing Signal Amplification (TSA), which is based on patented catalyzed reporter deposition (CARD). U.S. Patent No. 2 6,593,100, entitled “Enhanced catalyzed reporter deposition” reveals the intensification of the catalysis of an enzyme in a CARD or TSA method through the reaction of a labeled phenol conjugate with an enzyme, in which the reaction is carried out in the presence of a reagent intensification.
[005] Although methods such as those described above have been employed to increase the signals obtained from tests, the results of these methods indicate that the signal amplification is impaired by the corresponding background signal amplification. Thus, there is an ongoing need for signal amplification that can produce optimal results without a corresponding increase in background signals. BRIEF DESCRIPTION OF THE INVENTION
[006] The present disclosure deals with a method for the detection of a target in a sample, through proximal deposition of a marker, which comprises: making contact with the sample with a recognition solution, in which the recognition solution includes a specific binding portion, which is specific to the target; labeling the specific binding portion with an enzyme; put the sample in contact with a detection solution, where the detection solution comprises an enzymatic substrate so that the marker is deposited in the vicinity of the target in the presence of a deposition intensifier that has a formula:

[007] where R1, R2, R3 and R4 are independently selected from aliphatic, aryl, halogen, a portion containing heteroatom, and hydrogen; R1 and / or R3 can be linked to R2 to form a fused aromatic ring system; R5 is a portion that contains heteroatom; A is selected from a carbon atom, a heteroatom other than sulfur and any combination thereof; n is 1 to 5; and detect the marker. In reference to this method, placing the sample in contact with a detection solution may include enzymatically oxidizing the enzyme substrate through the use of an oxidizing agent to form the marker. In particular disclosed embodiments, enzymatically oxidizing the enzyme substrate through the use of an oxidizing agent comprises reducing the solubility or stability of the enzyme substrate so that the enzyme substrate becomes deposited as the marker.
[008] In particular revealed embodiments, the enzyme substrate is selected from the group consisting of a conjugate of tiramide and a chromogen and the deposition enhancer can have a formula,

[009] where R1, R2, R3θ R4 are selected from hydrogen and hydroxyl and each A is a carbon atom. In certain embodiments disclosed, R1, R3 and R4 are hydrogen and R2 is hydroxyl.
[010] Other examples of the deposition intensifier include those that have a formula,

[011] where R1, R2 and R3 are independently selected from alkyl, alkene, alkaline, hydrogen, iodine, bromine, chlorine, fluorine and combinations thereof.
[012] Particular revealed achievements deal with the use of an enzyme, which can be an oxidoreductase or a peroxidase. In addition, the enzyme can be selected from horseradish peroxidase, glutathione peroxidase, and microoxidase. The specific binding portion disclosed typically comprises an antibody or a nucleic acid.
[013] In reference to the disclosed method, the deposition of the marker in the vicinity of the target in the presence of the deposition intensifier includes the deposition intensifier at a concentration ranging from about 5 mM to about 15 mM.
[014] In particular revealed embodiments, the enzyme substrate can be selected from 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole, tetramethylbenzidine, a fluorescein, a luminophore, a coumarin, a BODIPY dye, a resorufin, rhodamine, or a derivative thereof. More typically, the enzyme substrate is a tyramine derivative.
[015] When the sample is brought into contact with a detection solution, it is typically exposed to the enzyme substrate in a concentration ranging from more than 0 mM to about 8 mM. In addition, the detection solution can additionally comprise an accelerator selected from a heteroaryl compound, a boronic acid, a phenolic compound, or a combination thereof. The heteroaryl compound can be selected from imidazole, L-histidine, pyridine A / -oxide, pyrimidine N-oxide, / V-methyl morpholine oxide, and 2,2,6,6-tetramethylpiperidine-l- oxila. In addition, the detection solution may additionally comprise a nonionic surfactant selected from a polyoxyethylene lauryl ether that has a formula (C2H4O) 23Ci2H25OH; polyoxyethylene (20) sorbitan monoalkylate, wherein the monoalkylate comprising between 8 and 14 carbons; a linear secondary alcohol polyoxyethylene having a formula Ci2-i4H25-2θO (CH2CH2θ] x, where x is equal to an integer between 2 and 12; and polyoxyethylene octyl phenyl ether. In particular revealed embodiments, the detection solution can additionally comprise an antioxidant selected from sodium bisulfate, sodium stannate, sodium metabisulfate and combinations thereof, and / or a salt containing Group I or Group II metal that has an MX2 or MX formula where M is a Group I or Group II metal selected from lithium, sodium, potassium, cesium, calcium, magnesium, strontium and barium, and X is selected from fluorine, chlorine, bromine, iodine, carbonate, hydroxide and phosphate.
[016] Also contemplated in the present disclosure is a composition to detect a target in a sample, through proximal deposition of a marker, which comprises a deposition intensifier that has a formula,

[017] where R1, R2, R3 and R4 are independently selected from aliphatic, aryl, halogen, a portion containing heteroatom, and hydrogen; R1 and / or R3 can be linked to R2 to form a fused aromatic ring system; R5 is a portion that contains heteroatom; A is selected from a carbon atom, a heteroatom other than sulfur and any combination thereof; n is 1 to 5; and an enzyme substrate.
[018] In certain embodiments revealed, the deposition intensifier has a formula,

[019] where R1, R2, R3 and R4 are selected from aliphatic, aryl, halogen, a portion that contains heteroatom, and hydrogen; R1 and / or R3 can be linked to R2 to form a fused aromatic ring system; A is selected from a heteroatom, other than sulfur, a carbon atom and combinations thereof. Typically, the deposition intensifier can have a concentration ranging from about 5 mM to about 15 mM and the enzyme substrate has a concentration ranging from more than 0 mM to about 8 mM, with the enzyme substrate being selected at from 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole, tetramethylbenzidine, a fluorescein, a luminophore, a coumarin, a BODIPY dye, a resorufin, a rhodamine, a tiramide, or a derivative thereof.
[020] In particular disclosed embodiments, the composition may additionally comprise an accelerator selected from a heteroaryl compound, a boronic acid, a phenolic compound, or a combination thereof; a nonionic surfactant selected from Brij® 35, TWEEN®, Tergitol ™ and Triton ™; and an antioxidant selected from sodium bisulfate, sodium stannate, sodium metabisulfate and combinations thereof.
[021] Also revealed is a kit comprising a detection solution, which comprises a deposition intensifier and an enzyme substrate, in which the deposition intensifier has a formula,

[022] where R1, R2, R3 and R4 are independently selected from aliphatic, aryl, halogen, a portion that contains heteroatom, and hydrogen; R1 and / or R3 can be linked to R2 to form a fused aromatic ring system; R5 is a portion that contains heteroatom; A is selected from a carbon atom, a heteroatom other than sulfur and any combination thereof; n is 1 to 5.
[023] In particular embodiments, the heteroaryl compound has a formula according to
and / or a formula according to
and / or a formula according to
wherein R8 to R22 are independently aliphatic, aryl, halogen, a portion containing heteroatom, hydrogen or any combination thereof; R23 is [O] 'or [O] -; A is a carbon atom, a heteroatom, different from sulfur or any combination thereof; B is oxygen, carbon, or nitrogen; em is 0 to 2. Halogen can be selected from iodine, bromine, chlorine or fluorine, and the portion containing heteroatom can be selected from hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate , phosphine, amide, NR6R7 where R6 and R7 are, independently, hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination thereof. In particular embodiments, each of R17 and R21 comprises a methyl group and is 2. Exemplary heteroaryl compounds include, but are not limited to, imidazole, L-histidine, pyridine A-oxide, pyrimidine A-oxide, pyrimidine oxide / V-methyl morpholine, and 2,2,6,6-tetramethylpiperidine-1-oxyl.
[024] In particular embodiments, the optional intensifier is a compound containing boron, such as an organic boronic acid. Exemplary organic boronic acids include, but are not limited to, boric acid.
[025] In particular embodiments, the optional intensifier is a phenolic compound that has a formula

[026] where R27, R28, R29, R30 and R31 can be selected independently from aliphatic, aryl, a portion containing heteroatom, hydrogen, or any combination thereof. The heteroatom-containing portion is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide and NR5R6, where R5 and R6 are independently: hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl and any combination of the same. In particular embodiments, any two adjacent groups selected from R27, R28, R29, R30 and R31 can be linked to form a non-aromatic, aromatic or fused ring system. Exemplary phenolic compounds include, but are not limited to, pyrocatechol.
[027] In particular embodiments, the current method may further comprise: immobilizing the enzyme / specific binding moiety conjugate on the target in the sample; contacting the sample with a solution comprising a tiramide / hapten conjugate; put the sample in contact with the intensification solution; put the sample in contact with the oxidizer; and locating the target in the sample by detecting the tiramide / hapten conjugate. In particular embodiments, the detection of the tiramide / hapten conjugate additionally comprises: placing the sample in contact with an anti-hapten antibody capable of recognizing and binding to the tiramide / hapten conjugate and a detectable portion capable of being detected by means of the use of sets of fluorescent or deposition procedures; and detecting the detectable portion. In particular embodiments, the tiramide / hapten conjugate comprises a hapten conjugated directly to tyramine or via a linker. Typically, the linker is aliphatic or heteroaliphatic.
[028] The previous advantages and resources of this disclosure will become more evident from the following detailed description, which follows in reference to the attached figures. BRIEF DESCRIPTION OF THE DRAWINGS
[029] Figure 1 is a digital image showing the IHC staining of bcl2 in amygdala tissue using a standard ultraView ™ Detection Kit.
[030] Figure 2 is a digital image showing the use of 10 mM imidazole as a base buffer in the diaminobenzidine staining solution (DAB) for IHC staining of bcl2 in amygdala tissue.
[031] Figure 3 is a graph showing the influence of 4-acetylamidophenyl boronic acid on the apparent Vmax for HRP oxidized DAB when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[032] Figure 4 is a graph showing the influence of imidazole on apparent Vmax for DAB oxidized by horseradish peroxidase (HRP) when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[033] Figure 5 is a graph showing the influence of L-histidine on apparent Vmax for HRP oxidized DAB when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[034] Figure 6 is a graph showing the influence of boric acid on apparent Vmax for HRP oxidized DAB when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[035] Figure 7 is a graph showing the influence of pyrimidine on apparent Vmax for HRP oxidized DAB when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[036] Figure 8 is a graph showing the influence of 2-hydroxypyrimidine on the apparent Vmax for HRP oxidized DAB when added to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[037] Figure 9 is a graph showing the influence of potential intensifiers on the apparent Vmax for HRP oxidized DAB when added sequentially to the ultraView ™ Detection Kit. The optical density of oxidized DAB was monitored at 455 nm.
[038] Figure 10 is a digital DAB staining image of the ultraView ™ of bcl2 tissue (amygdala) using a 10 mM imidazole based on the DAB chromogenic solution.
[039] Figure 11 is a digital DAB staining image of the ultraView ™ of bcl2 tissue using a 10 mM L-histidine based on the DAB chromogenic solution.
[040] Figure 12 is a digital DAB staining image of the ultraView ™ of bcl2 tissue using a 10 mM pyrimidine-A / -oxide in 10 mM L-histidine based on the DAB chromogenic solution. .
[041] Figure 13 is a digital DAB staining image of the ultraView ™ of bcl2 tissue using a 10 mM 2-hydroxypyrimidine in 10 mM L-histidine based on the DAB chromogenic solution.
[042] Figures 14 to 17 are digital images of IHC staining of bcl2 in amygdala tissue using a standard VMSI ultraView ™ Detection Kit with or without DAB "enhancing solutions". DAB intensification solutions: Figure 14: no intensification; Figure 15: 100 mM imidazole, 50 mM boric acid; Figure 16: 50 mM L-histidine, 10 mM pyrimidine; Figure 17: 10 mM L-histidine, 10 mM 2-hydroxypyridine, 10 mM calcium chlorine, 10 mM boric acid. Pathological score for signal / background was: Figure 14, 3.75 / 0.5; Figure 15, 4.0 / 0.75; Figure 16, 4 + / 0.5; Figure 17, 4 / 0.5.
[043] Figure 18 is a graph showing the influence of DAB chromogenic solutions of L-histidine and imidazole on the apparent Vmax for HRP oxidized DAB when combined with 10 mM pyrimidine. The optical density of oxidized DAB was monitored at 455 nm.
[044] Figure 19 is a graph showing the influence of chromogenic DAB solutions of L-histidine and imidazole on the apparent Vmax for HRP oxidized DAB when combined with 10 mM 2-hydroxypyrimidine, 10 mM boric acid and 10 mM of calcium chlorine. The optical density of oxidized DAB was monitored at 455 nm.
[045] Figure 20 is a graph showing the influence of enhancers on apparent Vmax for HRP oxidized DAB when combined with 50 mM imidazole, 10 mM calcium chlorine and 10 mM boric acid. The optical density of oxidized DAB was monitored at 455 nm.
[046] Figure 21 is a digital image of ISH staining of HER-2 probe DAB in HER-2 3 x 1 mouse xenographs of HER-2 positive CaLu3 carcinoma cell lines as the UltraView ™ detection.
[047] Figure 22 is a digital image of ISH staining of HER-2 probe DAB in 3 x 1 HER-2 mouse xenographs of HER-2 positive CaLu3 carcinoma cell lines with a chromogenic solution of DAB and intensification with 10 mM of L-histidine.
[048] Figures 23 to 26 are digital images of the IHC staining of bcl2 in amygdala tissue using the tiramide amplification system with and without intensification of HRP oxidation for both deposition of DAB and tiramide. Figure 23: no intensification for deposition of DAB or tiramide; Figure 24: no deposition of intensification tiramide, deposition of intensification DAB; Figure 25: deposition of intensification tiramide, no deposition of intensification DAB; Figure 26: deposition of DAB and intensification tiramide.
[049] Figures 27 and 28 are digital images of IHC staining of bcl2 in amygdala tissue using the tiramide amplification system for tyramide deposition with 10 mM 2-hydroxypyrimidine (Figure 27) and without oxidation intensification of HRP (Figure 28).
[050] Figure 29 is a digital image of ISH staining of the ribsome 18s in CaLu-3 xenograft tissues with ribsound 18s, and a DAB enhanced with both 10 mM 2-hydroxypyridine and 10 mM L-histidine.
[051] Figure 30 is a digital image of ISH staining of ribsome 18s in CaLu-3 xenograft tissues with ribsound 18s and no enhancement.
[052] Figure 31 is an IHC staining digital image of HPV in CaSki xenograft tissues with haptenylated HP probe, and an DAB enhanced with both 10 mM 2-hydroxypyridine and 10 mM L-histidine.
[053] Figure 32 is a digital image of IHC staining of HPV in CaSki xenograft tissues with haptenylated HPV probe and no enhancement.
[054] Figure 33 is a digital image of IHC staining of HPV in HeLa xenograft tissues with haptenylated HP probe, and a DAB enhanced with both 10 mM 2-hydroxypyridine and 10 mM L-histidine.
[055] Figure 34 is a digital image of IHC staining of HPV in HeLa xenograft tissues with haptenylated HPV probe and no enhancement.
[056] Figure 35 is a digital image of IHC staining of HPV in C33 xenograft tissues with haptenylated HPV probe, and an DAB enhanced with both 10 mM 2-hydroxypyridine and 10 mM L-histidine.
[057] Figure 36 is a digital image of IHC staining of HPV in C33 xenograft tissues with haptenylated HPV probe and no enhancement.
[058] Figure 37 is a digital image of IHC staining of CD20 in amygdala tissues with anti-CD20 probe, and DAB enhanced with both 10 mM 2-hydroxypyridine and 10 mM L-histidine.
[059] Figure 38 is a digital image of IHC staining of CD20 in tonsil tissues with anti-CD20 probe and no enhancement.
[060] Figure 39 is a digital image of IHC staining of CD20 in amygdala tissues with anti-CD20 probe, and enhanced ECA deposition with 50 mM L-histidine and 10 mM 2-hydroxypyridine.
[061] Figure 40 is a digital image of IHC staining of CD20 in tonsil tissues with anti-CD20 probe and no enhancement.
[062] Figure 41 is a digital image of IHC staining of Ki67 in amygdala tissues with anti-Ki67 probe, and enhanced AEC deposition with 50 mM L-histidine and 10 mM 2-hydroxypyridine.
[063] Figure 42 is a digital image of IHC staining of Ki67 in tonsil tissues with anti-Ki67 probe and no enhancement.
[064] Figures 43 to 46 are digital images of the IHC staining of bcl2 in amygdala tissue through the use of an amplification system of HRP oxidation with and without intensification for both deposition of DAB and withdrawal. Figure 43: no intensification for deposition of DAB or take away; Figure 44: no deposition of intensification take-off, deposition of intensification DAB; Figure 45: deposition of intensification take-off, no deposition of intensification DAB; Figure 46: deposition of DAB and intensification. DETAILED DESCRIPTION OF THE INVENTION
[065] I. Introduction
[066] Diseases such as cancer can be diagnosed using several different methods. One method is to identify the presence of a biological marker, such as a biological cancer marker, in tissue or cells, in which the biological marker is correlated, or considered to be correlated, with a particular type of cancer. Immunohistochemistry is often used to target biological protein markers that are associated with a particular type of cancer, while sets of in situ hybridization procedures are often employed to target nucleic acid sequences that are associated with a particular type of cancer.
[067] In situ hybridization and immunohistochemistry methods to target identification have become increasingly important in research applications and for physicians, for example, for diagnostic and / or prognostic purposes. However, these sets of procedures may be limited by the detectable signal emitted by a detection portion that interacts with or is deposited on the target molecule present or that is considered to be present in a tissue sample, such as a protein and / or a target molecule of nucleic acid. Theoretically, one way to increase the signal obtained is to increase the deposition of a detectable portion on the target molecule, for example, by increasing the deposition rate, so that a larger signal can be obtained in a shorter amount of time.
[068] As revealed in this document, an innovative formulation of a DAB chromogen acts synergistically to provide maximized DAB deposition during ISH or IHC tissue staining. The innovative formulation of the DAB chromogen uses an organic intensifier as a buffer salt in combination with a variety of non-organic / organic intensifiers and surfactant to synergistically maximize the deposition of DAB and therefore the signal. Also disclosed in this document are methods of using the formulations disclosed to enhance tissue staining of ISH and / or IHC.
[069] The methods as described in this document find utility for diagnostics, in which results provided by the disclosed methods are used not only for diagnosis, but also to determine optimal treatment, and to track the progress and success of such treatment, in an environment clinical.
[070] II. Terms
[071] Unless otherwise stated, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995; and George P. Rédei, Encyclopedic Dictionary of Genetics, Genomics, and Proteomics, 2nd Edition, 2003.
[072] The singular forms "one", "one", "o" and "a" refer to one or more of one, unless the context clearly indicates otherwise. For example, the term "which comprises a cell" includes single or plural cells and is considered equivalent to the expression "which comprises at least one cell." The term "or" refers to the single element of alternative elements mentioned or a combination of two or more elements, unless the context clearly indicates otherwise. A wavy line (“- ~ w”) is used to indicate a bond disconnection, and a dashed line is used to illustrate that a bond can be formed at a particular position.
[073] Although methods and materials similar or equivalent to those described in this document can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting.
[074] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art to practicing the present disclosure.
[075] Aliphatics: Portions that include cycloalkyl, alkyl, alkenyl, alkynyl and halogenated groups. A "lower aliphatic" group is a branched or unbranched aliphatic group that has 1 to 10 carbon atoms. This term encompasses substituted aliphatic compounds, saturated aliphatic compounds and unsaturated aliphatic compounds.
[076] Alkyl: An unbranched or branched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl , decila, tetradecila, hexadecila, eicosila, tetracosila and the like. A "lower alkyl" group is an unbranched or branched saturated hydrocarbon that has 1 to 10 carbon atoms. The terms "halogenated alkyl" or "haloalkyl group" refer to an alkyl group as defined above with one or more hydrogen atoms present in these groups replaced with a halogen (F, Cl, Br, I). The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopeptyl, cyclohexyl, etc.
[077] The term "heterocycloalkyl group" is a cycloalkyl group in which at least one of the carbon atoms in the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur or phosphorus. Optionally substituted groups, such as "substituted alkyl", describe groups, such as an alkyl group, which have 1 to 5 substituents, typically 1 to 3 substituents, selected from alkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy , amino, aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, optionally substituted heteroaryl, optionally substituted heterocyclyl, hydroxy, thiol and thioalkoxy.
[078] Amplification: Amplification refers to the act or result of creating a stronger signal. The amplification can be an increase in the magnitude of the signal and / or an increase in the signal in relation to the background, for example, increased signal / noise ratio.
[079] Antibody: Collectively refers to immunoglobulins or immunoglobulin-like molecules (which include, by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103 M'1 greater, at least 104 M-1 greater or at least 105 M-1 greater than a constant binding to other molecules in a biological sample).
[080] More particularly, "antibody" refers to a polypeptide linker that comprises at least one variable region of light chain or heavy chain immunoglobulin that specifically recognizes and binds to an epitope of an antigen. Antibodies can be composed of a light chain and a heavy chain, each of which has a variable region, called the variable heavy region (VH) and the variable light region (VL). Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
[081] Antibodies include intact immunoglobulins and variants and portions thereof well known in the art. Antibody fragments include proteolytic antibody fragments [such as F (ab ') 2 fragments, Fab' fragments, Fab'-SH fragments and Fab fragments as are known in the art], recombinant antibody fragments (such as sFv fragments, dsFv fragments , bispecific sFv fragments, bispecific dsFv fragments, F (ab) '2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies and triabodies (as are known in the art), and camelid antibodies (see, for example, US Patent No. 6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808). A scFv protein is a fusion protein in which an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region are linked by a linker, whereas in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. still way are genetically engineered such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994 to 1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Edition, W.H. Freeman & Co., New York, 1997.
[082] Typically, a naturally occurring immunoglobulin has heavy chains and light chains interconnected by disulfide bonding. There are two types of light chain, lambda and cap. There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.
[083] Each light and heavy chain contains a constant region and a variable region; regions are also known as "domains." In combination, the variable regions of the light and heavy chain specifically bind the antigen. Heavy and light chain variable regions contain a "human support" region interrupted by three hypervariable regions, also called "complementarity determination regions" or "CDRs." The extent of the human support region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is now maintained online. Human support region sequences from different heavy or light chains are relatively conserved in one species. The human support region of an antibody, which is the combined human support region of the compound heavy and light chains, serves to position and align the CDRs in a three-dimensional space.
[084] CDRs are primarily responsible for binding to bind to an epitope on an antigen. The CDRs of each chain are typically called CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminal, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the antibody heavy chain in which it is found, while a VL CDR1 is CDR1 in the variable domain of the antibody light chain in which it is found.
[085] Antigen: A molecule that enhances an immune response. Antigens are usually proteins or polysaccharides. An epitope is an antigenic determinant composed of chemical groups or peptide sequences in a molecule that obtains a specific immune response. An antibody binds to a particular epitope or antigen. The binding of an antibody to a particular epitope or antigen of an antigen can be used to locate the position of the antigen, for example, within or on a biological sample, or to determine whether the particular antigen is present in a biological sample. An antigen of interest is an antigen that an HCI assay is designed to detect in a test sample. For example, to detect an antigen of interest, the primary antibody used in the IHC assay specifically binds to the antigen of interest.
[086] An epitope is a site on a target molecule (for example, an antigen, such as a nucleic acid or protein molecule) to which an antigen-binding molecule (for example, an antibody, antibody fragment, protein, scaffold containing antibody-binding regions, or aptamer) binds. Epitopes can be formed from both juxtaposed and contiguous non-contiguous residues (for example, amino acids or nucleotides) of the target molecule (for example, a protein / protein interface). Epitopes formed from contiguous residues (e.g., amino acids or nucleotides) are typically contained in exposure to denaturing solvents while epitopes formed through tertiary folding are typically lost in treatment with denaturing solvents. An epitope typically includes at least 3 and, more commonly, at least 5 or 8 10 residues (for example, amino acids or nucleotides). Typically, an epitope is also less than 20 residues (for example, amino acids or nucleotides) in length, such as less than 15 residues or less than 12 residues.
[087] Aromatic: A term that describes conjugated rings that have unsaturated bonds, isolated electron pair or empty orbitals, that exhibit stronger stabilization than would be expected by conjugation stabilization alone. It can also be considered a manifestation of cyclical displacement and resonance.
[088] Aryl: An aromatic compound substantially based on hydrocarbon, or a radical thereof (eg, CeHs) as a substituent attached to another group, particularly other organic groups, which has a ring structure as exemplified by benzene, naphthalene, phenanthrene, anthracene, etc. This term also includes substituted aryl compounds.
[089] Arylalkyl: A compound, or a radical thereof (C7H7 for toluene) as a substituent attached to another group, particularly other organic groups, which contains both aliphatic and aromatic structures.
[090] Binding or stable binding: An association between two substances or molecules, such as the association of a specific portion or binding agent (eg, antibody) with an antigen.
[091] Binding affinity: The tendency of a molecule to bind (typically non-covalently) to another molecule, such as the tendency from one member of a specific binding pair to another member of a specific binding pair. A binding affinity can be measured as a binding constant, whose binding affinity for a specific binding pair (such as an antibody / antigen pair or nucleic acid probe / nucleic acid sequence pair) can be at least 1 x 105 M-1, such as at least 1 x 106 M-1, at least 1 x 107 M-1 or at least 1 x 108 M_ 1. In one embodiment, the binding affinity is calculated by modifying the Scatchard method described by Frankel et al., Mol. Immunol., 16: 101 to 106.1979. In another embodiment, binding affinity is measured using an antigen / antibody dissociation rate. In yet another embodiment, a high binding affinity is measured using a competition radioimmunoassay. In several examples, a high binding affinity for an antibody / antigen pair is at least about 1 x 108 M'1. In other embodiments, a high binding affinity is at least about 1.5 x 108 M-1, at least about 2.0 x 108 M'1, at least about 2.5 x 108 M'1, at least at least about 3.0 x 108 M'1, at least about 3.5 x 108 M'1, at least about 4.0 x 108 M'1, at least about 4.5 x 108 M-1 , or at least about 5.0 x 108 M-1.
[092] Chromogen: A substance capable of conversion to a colored product, such as a pigment or dye. Certain chromogens are electron donors that, when oxidized, become a colored product. The production of a colored product, and the property of becoming insoluble by chemical conversion, such as oxidation, makes chromogens useful for IHC. Particular examples of chromogenic compounds, without limitation, include diaminobenzidine (DAB), tetramethylbenzidine (TMB), 2,2'-azino-di- [3-ethylbenzothiazoline sulfonate] (ABTS), iodonitrotetrazolium (INT), tetrazolium blue and violet tetrazolium.
[093] DAB is a chromogen that produces a brown end product that is highly insoluble in alcohol and other organic solvents. In some examples, DAB is the substrate for an enzyme, such as HRP.
[094] Sufficient conditions for detection: Any environment that allows the desired activity, for example, that allows a probe to connect to a target and that the interaction is detected. For example, such conditions include appropriate temperatures, buffer solutions and means of detection such as microscopes and digital imaging equipment.
[095] Contact: Positioning that allows the association between two or more portions, particularly direct physical association, for example, both in solid and / or in liquid form (for example, a positioning of a biological sample, such as a biological sample attached to a conductor, in contact with a composition, such as a solution containing the compositions disclosed in this document).
[096] Control: A sample or procedure performed to assess the validity of the test. In one example, a control is a quality control, just like a positive control. For example, a positive control is a procedure or sample, such as a tissue or cell, which is similar to the actual test sample, but which is known from previous experience to offer a positive result. A positive control confirms that the basic test conditions produce a positive result, even if none of the actual test samples produce such a result. In a particular example, a positive control is a sample known through previous testing to contain the suspected antigen.
[097] In other examples, a control is a negative control. A negative control is a procedure or test sample known from previous experience for offering a negative result. The negative control shows the baseline result obtained when a test does not produce a measurable positive result; the negative control value is often treated as a "background" value to be subtracted from the test sample results. In a particular example, a negative control is a reagent that does not include the specific primary antibody. Other examples include calibrator controls, which are samples that contain a known amount of a control antigen. Such control gauges have an expected signal strength, and therefore can be used to correct color variability between or within operation.
[098] Conjugate: A molecule that comprises two independent molecules, which have been joined through a bond (typically an ionic or covalent bond). In some instances, a specific binding agent or portion is conjugated to an enzyme that acts on a substrate to produce a detectable portion or label.
[099] Conjugation, union, connection or connection: Unite one molecule to another molecule to create a larger molecule. For example, making two polypeptides into a contiguous polypeptide molecule, or covalently attaching a hapten or other molecule to a polypeptide, such as an scFv antibody. A bond can be by recombinant or chemical means. "Chemical medium" refers to a reaction between the portion of the antibody and the effector molecule so that there is a covalent bond formed between the two molecules to form a molecule.
[100] Coupled: The term "coupled" means joined, directly or indirectly. A first atom or molecule can be directly coupled or indirectly coupled to a second atom or molecule. A secondary antibody provides an example of indirect coupling. A specific example of indirect coupling is a primary rabbit anti-hapten antibody that is linked by a mouse anti-rabbit IgG antibody, which is in turn linked by a goat anti-mouse IgG antibody that is covalently linked to a detectable tag.
[101] Derivative: In chemistry, a derivative is a compound that is derived from a similar compound or a compound that can be thought of as arising from another compound, for example, if an atom is replaced with another atom or group of atoms. The latter definition is common in organic chemistry. In biochemistry, the word is used for compounds that at least theoretically can be formed from the precursor compound.
[102] Detectable Tag: A molecule or material that can produce a detectable signal (such as visually, electronically or otherwise) that indicates the presence and / or concentration of a target, such as a target molecule, in a sample, such like a tissue sample. When conjugated to a specific binding molecule, the detectable tag can be used to locate and / or quantify the target to which the specific binding molecule is targeted. In this way, the presence and / or concentration of the target in a sample can be detected by detecting the signal produced by the detectable tag. A detectable tag can be detected directly or indirectly, and several different detectable tags conjugated to different specific binding molecules can be used in combination to detect one or more targets. For example, a first detectable tag, such as a hapten conjugated to a target specific antibody, can be detected indirectly through the use of a second detectable tag that is conjugated to a molecule that specifically binds to the first detectable tag. Multiple detectable tags that can be separately detected can be conjugated to different specific binding molecules that specifically bind to different targets to provide a multiplexed assay that can provide detection of multiple targets in a sample.
[103] Discoverable labels include colored, fluorescent, phosphorescent and luminescent materials and molecules, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of a sample), haptens that can be detected through antibody / hapten binding interaction through the use of additional detectably labeled antibody conjugates, and magnetic and paramagnetic materials or molecules. Particular examples of detectable tags include: enzymes, such as horseradish peroxidase, glucose oxidase, β-galactosidase or β-glucuronidase; fluorophores (many additional examples of fluorescent molecules can be found in The Handbook - A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR); nanoparticles, such as quantum dots (for example, U.S. Patent Nos. 6,815,064, 6,682596 and 6,649,138); metal chelates, such as DPTA and DOTA chelates of paramagnetic or radioactive metal ions such as Gd3 +; chromogens; and liposomes, for example, liposomes that contain trapped fluorescent molecules.
[104] Where the detectable tag includes an enzyme, a detectable substrate such as a chromogen, a fluorogenic compound or a luminogenic compound is used in combination with the enzyme to generate a detectable signal (a wide variety of such compounds are commercially available, for example. example from Life Technologies, Carlsbad, CA).
[105] Alternatively, an enzyme can be used in a metallographic detection scheme. Metallographic detection methods include using an enzyme in combination with a water-soluble metal ion and an inactive substrate for oxide-reduction of the enzyme. The substrate is converted into an active agent for oxide-reduction by the enzyme, and the active agent for oxide-reduction reduces the metal ion, causing it to form a detectable precipitate. (See, for example, Copending Patent Application No. U.S. Series No. 11 / 015,646, filed December 20, 2004, PCT Application No. 2005/003777 and Patent Application Publication No. U.S. 2004/0265922). Metallographic detection methods include using an oxidoreductase enzyme (such as horseradish peroxidase (HRP)) together with a water-soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate (see, for example, for example, U.S. Patent No. 6,670,113).
[106] Detergent or Surfactant: A substance that reduces the surface tension of water. Specifically, a detergent or surfactant is an active surface agent, or surfactant, that concentrates on oil / water interfaces and exerts an emulsifying action. Detergents are classified as anionic, cationic or non-ionic, depending on their mode of chemical action. Non-ionic detergents work through a hydrogen bonding mechanism. In addition, surfactants or detergents reduce the interfacial tension between two liquids. A surfactant molecule typically has an ionic or polar "head" and a non-polar hydrocarbon "tail". Upon dissolution in water, the surfactant molecules aggregate and form micelles, in which the non-polar tails are oriented inwards and the ionic or polar heads are oriented outwards towards the aqueous environment. The non-polar tails create a non-polar "pocket" in the micelle. Non-polar compounds in the solution are sequestered in the pockets formed by the surfactant molecules, thus allowing the non-polar compounds to remain mixed in the aqueous solution.
[107] Detection: To determine whether an agent (such as a particular signal or antigen, protein or nucleic acid) is present or absent, for example, in a sample. In some examples, this may additionally include quantification and / or location, for example, location in a particular cell or cell compartment. “Detect” refers to any method of determining whether or not something exists, such as determining whether a target molecule is present in a biological sample. For example, "detect" may include using a mechanical or visual device to determine whether a sample exhibits a specific characteristic. In certain examples, detection refers to visually observing a probe attached to a target, or observing that a probe does not attach to a target. For example, light microscopy and other microscopic means are commonly used to detect chromogenic precipitates for methods described here.
[108] Electromagnetic radiation: Various electromagnetic waves that are propagated through periodic simultaneous variations in magnetic and electric field strength, which include radio waves, infrared, visible light, ultraviolet light, X-rays and gamma rays. In particular examples, electromagnetic radiation is emitted through a laser, which can have properties of monochromaticity, directionality, coherence, polarization and intensity.
[109] Emission or emission signal: Light of a particular wavelength generated from a source. In particular examples, an emission signal is emitted from a fluorophore after the fluorophore absorbs light at its excitation wavelength (s).
[110] Intensification (intensifier / intensification / intensification): An intensifier or intensification reagent is any compound or any combination of compounds sufficient to increase the catalytic activity of an enzyme, compared to the enzyme activity without such / such compound ( s). Intensifier (s) or intensifying reagent (s) can also be defined as a compound or combination of compounds that increases or accelerates the rate of binding of an activated conjugate to a receptor site. Stepping up (stepping up / stepping up) is a process by which the catalytic activity of an enzyme is increased by means of an intensifier, compared to a process that does not include such an intensifier. Intensifying (intensification / intensification) can also be defined as increasing or accelerating the rate of binding of an activated conjugate to a receptor site. Stepping up (stepping up / stepping up) can be measured visually, such as by scoring by a pathologist. In particular achievements, scores range from more than 0 to more than 4, with the highest number indicating better visual detection. More typically, scores range from more than 0 to about 4 ++, such as 1, 1.5, 2, 2.5, 3, 3.5, 3.75, 4, 4+ and 4 ++. In addition, intensification (intensification / intensification) can be measured by determining the apparent Vmax of an enzyme. In particular embodiments, the term encompasses apparent Vmax values (measured as optical density / minute) ranging from more than 0 mOD / min to about 400 mOD / min, such as about 15 mOD / min, 18 mOD / min, about 20 mOD / min, about 40 mOD / min, about 60 mOD / min, about 80 mOD / min, about 100 mOD / min, about 120 mOD / min, about 140 mOD / min, about 160 mOD / min, about 200 mOD / min, about 250 mOD / min, about 300 mOD / min, about 350 mOD / min and about 400 mOD / min. More typically, Vmax ranges from more than 0 mOD / min to about 160 mOD / min, such as about 20 mOD / min, about 40 mOD / min, about 60 mOD / min, about 80 mOD / min , about 100 mOD / min, about 120 mOD / min, about 140 mOD / min and about 160 mOD / min. In addition, intensification can occur through the use of any intensifier concentration greater than 0 mM. Typically, intensification occurs at intensifier concentrations ranging from more than 0 mM to about 100 mM; even more typically from about 0.01 mM to about 100 mM, such as about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.10 mM, about 0, 20 mM, about 0.50 mM, about 1.0 mM, about 2.0 mM, about 3.0 mM, about 5.0 mM, about 10.0 mM, about 20.0 mM, about 30.0 mM, about 40.0 mM, about 50.0 mM, about 75.0 mM, or about 100.0 mM, such as about 0.01 mM to about 0 , 10 mM, about 0.05 mM to about 0.50 mM, about 0.4 mM to about 1.0 mM, about 0.5 mM to about 2.0 mM, about 1, 0 mM to about 10.0 mM, about 5.0 mM to about 50.0 mM, and about 20.0 mM to about 100.0 mM.
[111] Excitation or excitation signal: Light of a particular wavelength required and / or sufficient to excite an electron transition to a higher energy level. In particular examples, an excitation is light of a particular wavelength needed and / or sufficient to excite a fluorophore to a state such that the fluorophore emits a wavelength of light different (such as longer) from the wavelength excitation signal light.
[112] Fixation: A process that preserves tissue and cell constituents as close to a life-like state as possible and allows them to undergo preparatory procedures without alteration. Fixation suspends the bacterial decomposition and autolysis processes that start with the death of the cell, and stabilizes the tissue and cellular constituents so that they resist the subsequent stages of tissue processing, such as for IHC.
[113] Tissues can be fixed by perfusion with or submerged in a fixative, such as an aldehyde (such as formaldehyde, paraformaldehyde, glutaraldehyde and the like). Other fixatives include oxidizing agents (for example, complexes and metal ions, such as osmium tetroxide and chromic acid), protein denaturing agents (for example, acetic acid, methanol and ethanol), fixators of unknown mechanism (for example, mercuric chlorine, acetone and picric acid), combination reagents (for example, Carnoy fixative, metacarno, Bouin fluid, B5 fixative, Rossman fluid and Gendre fluid), microwaves and miscellaneous (for example, volume fixation excluded and steam fixation). Additives can also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (eg, zinc chlorine, zinc sulfate and lithium salts), and lanthanum.
[114] The fixative most commonly used in sample preparation for HCI is formaldehyde, usually in the form of a formalin solution (4% formaldehyde in a buffer solution, called 10% buffered formalin).
[115] Fluorescence: A type of luminescence in which an atom or molecule absorbs energy and then emits visible light as it undergoes a transition from a lower to a higher electronic state. The term "fluorescence" is restricted to phenomena in which the time between absorption and energy emission is extremely short.
[116] Fluorescence in situ hybridization (FISH): FISH is a technique used to detect and locate the presence or absence of specific DNA and / or RNA sequences on chromosomes. FISH uses fluorescently labeled probes that connect only those parts of the chromosome with which they show a high degree of sequence similarity under defined reaction conditions. FISH can also be used to detect particular mRNA sequences in tissue samples.
[117] Fluorophore: A chemical compound that, when excited through exposure to a particular intensification such as a defined wavelength of light, emits light (fluoresces), for example, at a different wavelength (such as a wavelength) longer light wave).
[118] Fluorophores are part of the larger class of luminescent compounds. Luminescent compounds include chemiluminescent molecules, which do not require a particular wavelength of light to luminescent, but instead use a chemical source of energy. Therefore, the use of chemiluminescent molecules (such as equorine) eliminates the need for an external source of electromagnetic radiation, such as a laser.
[119] Examples of particular fluorophores that can be used in the probes disclosed in this document are provided in Patent No. 2 U.S. 5,866,366 to Nazarenko et al., Such as 4-acetamide-4'-isothiocyanatestilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1 acid -sulfonic (EDANS), 4-amino-N- [3-vinylsulfonyl) phenyl] naphthalimide-3,5 (Lucifer Yellow VS), N- (4-anilino-1-naphthyl) maleimide, anthranilamide, Bright Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcularine (Cumarana 151); cyanosine; 4 ', 6-diaminidino-2-phenylindole (DAPI); 5 ', 5 "-dibromopyrogalol-sulfonaphthalene (Bromopyrogalol Red); 7-diethylamino-3- (4'-isothiocyanatophenyl) -4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stylbene-2,2' -disulfonic; 4,4'-diisothiocyanato-stilbene-2,2'- disulfonic acid; 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dapilyl chlorine); 4-dimethylaminophenylazofeπyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiociaπate; erythrosine and derivatives such as erythrosine B and erythrosine isothiociaπate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiociaπate (FITC), and QFITC (XRITC); fluorescami; IR144; IR1446; Malachite; 4-methylumbelliferone; ortho-cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthalialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (CIBACRON ™ Bright Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chlorine, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiociaπate, sulforodamine B , sulforodamine 101 and sulforodamine 101 sulfonyl chlorine derivative (Texas Red); N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiociaπate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; LuzCycler Red 640; Cy5.5; and Cy5.
[120] Haptene: A molecule, typically a small molecule that can be specifically combined with an antibody, but is typically substantially incapable of being immunogenic other than in combination with an excipient molecule. Examples of haptens include, but are not limited to, fluorescein, biotin, nitroaryls, which include, but are not limited to, dinitrophenol (DNP), digoxigenin, oxazole, pyrazole, thiazole, benzofuran, triperpene, urea, thiourea, rotenoid, coumarin and cyclolignan .
[121] Heterobifunctional: Crosslinking agents contain at least two different reactive groups at each end, which are reactive towards numerous groups, including, but not limited to, sulfhydryl and amines, and create chemical covalent bonds between two or more molecules, for example, between a specific binding agent or portion (such as an antibody) and an enzyme (such as HRP).
[122] Hybridization: To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thus forming a duplex molecule. Hybridization conditions that result in particular degrees of stringency will vary depending on the nature of the hybridization method and the composition and length of the hybridization nucleic acid sequences. Generally, the hybridization temperature and ionic strength (such as the Na + concentration) of the hybridization buffer will determine the hybridization stringency. Calculations regarding hybridization conditions to achieve particular degrees of strictness are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11).
[123] Immunohistochemistry (IHC): A method of determining the presence or distribution of an antigen in a sample by detecting the interaction of the antigen with a specific portion or binding agent, such as an antibody. A sample that includes an antigen (such as a target antigen) is incubated with an antibody under conditions that allow an antibody / antigen binding. An antibody / antigen binding can be detected by means of a detectable tag conjugated to the antibody (direct detection) or by a detectable tag conjugated to a secondary antibody, which is raised against the primary antibody (for example, indirect detection). Detectable tags include, but are not limited to, radioactive isotopes, fluorochromes (such as fluorescein, fluorescein isothiocyanate and rhodamine), enzymes and chromogenic molecules.
[124] In situ hybridization (ISH): A type of hybridization that uses a labeled complementary DNA or RNA strand (ie probe) to locate a specific DNA or RNA sequence in a tissue portion or section (in situ) or, if the tissue is small enough (for example, plant seeds, Drosophila embryos), throughout the tissue (whole mount ISH). This is distinct from immunohistochemistry, which locates proteins in sections of tissue. DNA ISH can be used to determine chromosome structure, such as for use in medical diagnostics to assess chromosomal integrity. ISH by RNA (hybridization histochemistry) is used to measure and locate mRNAs and other transcripts in tissue or whole mount sections.
[125] For hybridization histochemistry, sample tissues and cells are usually treated to fix the target transcripts in place and to increase the probe's access to the target molecule. As mentioned above, the probe is complementary DNA or labeled complementary RNA (Riboseprobe). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed (after previous hydrolysis using RNase in the case of unhybridized excess RNA probe). Solution parameters, such as temperature, detergent and / or salt concentration, can be manipulated to remove any non-identical interactions (that is, only exact sequence correspondents will remain linked). Then, the labeled probe that has been labeled effectively, such as with radiolabeled, fluorescent or antigen-based bases (eg, digoxigenin), is located and potentially quantized in the tissue through the use of autoradiography, fluorescence microscopy or immunohistochemistry, respectively.
[126] Ligand: As used in this document, a ligand is a molecule or group of atoms positioned between two portions. Typically, linkers are bifunctional, that is, the linker includes a functional group at each end, where the functional groups are used to couple the linker to the two portions. The two functional groups can be the same, that is, a homobifunctional ligand, or different, that is, a heterofunctional ligand.
[127] Ligand peptide: A peptide in an antibody binding fragment (such as an Fv fragment) that serves to indirectly link the variable heavy chain to the variable light chain. "Ligand" can also refer to a peptide that serves to bind a target portion, such as a scFv, to an effector molecule, such as a cytotoxin or a detectable tag.
[128] Molecule of interest or Target molecule: A molecule for which the presence, location and / or concentration must be determined. Examples of molecules of interest include proteins and nucleic acid sequences present in tissue samples.
[129] Multiplex, -ado, -action: Accomplishments of the present disclosure allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, through the use of different plural conjugates. Multiplexing can include the identification and / or quantification of nucleic acids, usually DNA, RNA, peptides, proteins, both individually and in any and all combinations. Multiplexing can also include the detection of two or more of a gene, a messenger and a protein in a cell in its anatomical context.
[130] Neoplasm and Tumor: The process of uncontrolled and abnormal cell growth. Neoplasia is an example of a proliferative disorder.
[131] The neoplasm product is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that is not metastasized is called "benign." A tumor that invades the surrounding tissue and / or can metastasize is called "malignant." Examples of hematological tumors include leukemias, which include acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute and myeloblastic, myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia leukemia), chronic leukemias (such as chronic myelocytic leukemia) chronic myeloid and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (high-grade and indolent forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hair cell leukemia and myelodysplasia.
[132] Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, cancer of the colon, malignant lining of the colon, malignancy , breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, sebaceous gland carcinoma pheochromocytomas, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniofariogioma, ependymoma, pinealoma, hemangioblastoma , acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma).
[133] Oligonucleotide: A plurality of nucleotides joined together by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to portions that function similarly to oligonucleotides but have portions that do not occur naturally. For example, oligonucleotide analogs can contain non-naturally occurring moieties, such as altered sugar moieties or bonds between sugars, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid molecules.
[134] Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example, a sequence (such as DNA or RNA) that is at least 6 bases long, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases, or from about 6 to about 50 bases, for example, about 10 to 25 bases, such as 12, 15 or 20 bases.
[135] Probe: An isolated nucleic acid, an isolated synthetic oligonucleotide, attached to a detectable tag or reporter molecule. Typical labels include radioactive isotopes, enzyme substrates, cofactors, binders, fluorescent or chemiluminescent agents, haptens and enzymes. Methods for marking and guiding in choosing appropriate labels for various purposes are discussed, for example, in Sambrooketal. (In Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, Greene Publ. Assoc, and Wiley-Intertersciences, 1992).
[136] An individual of ordinary skill in the art will observe that the specificity of a particular probe increases with its length. Thus, the probes can be selected to provide a desired specificity, and can comprise at least 17, 20, 23, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of the desired nucleotide sequence. In particular examples, the probes can be at least 100, 250, 500, 600 or 1,000 consecutive nucleic acids of a desired nucleotide sequence.
[137] Polypeptide: A polymer in which monomers are amino acid residues that are joined through amide bonds. When amino acids are alpha-amino acids, the L-optical isomer or the D-optical isomer can be used. The terms "polypeptide" or "protein" as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to encompass naturally occurring proteins, as well as those that are produced synthetically or recombinantly.
[138] The term "residue" or "amino acid residue" includes the reference to an amino acid that is incorporated into a protein, polypeptide or peptide.
[139] Sample: The term "sample" refers to any solid, semi-solid or liquid substance (or material) in or in which a target may be present. In particular, a sample can be a biological sample or a sample obtained from a biological material. Examples of biological samples include tissue samples and cytological samples. In some instances, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any sample of fluid or solid obtained from, excreted by or secreted by any living organism, including, without limitation, single-celled organisms, such as bacteria, yeast, protozoa and amoebae, among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human individual or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained, for example, from blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, vitreous or aqueous humor, or any body secretion, a transudate, an exudate ( for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by a disease). A biological sample may furthermore be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or may include a cell (a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some instances, a biological sample is a nuclear extract. In some instances, a biological sample is bacterial cytoplasm. In other examples, a sample is a test sample. For example, a test sample is a cell, tissue or cell pellet section prepared from a biological sample obtained from an individual. In one example, the individual is someone who is at risk of acquiring or has acquired a particular condition or illness.
[140] Bind specifically: A term that refers to the binding of an agent that preferably binds to a defined target (such as an antibody to a specific antigen or a nucleic acid probe to a specific nucleic acid sequence). In relation to an antigen, "specifically bind" refers to the preferential association of an antibody or other ligand, completely or partially, with a specific polypeptide. In relation to a nucleic acid sequence, "specifically bind" refers to the preferred association of a nucleic acid probe, completely or partially, with a specific nucleic acid sequence.
[141] A specific moiety or binding agent binds substantially only to a defined target. It is recognized that a lesser degree of non-specific interaction can occur between a molecule, such as a specific moiety or binding agent, and a non-target nucleic acid or non-target polypeptide sequence. Although a selectively reactive antibody binds to an antigen, it can do so with low affinity. Specific antibody binding to antigen typically results in an increase of more than 2 folds, such as greater than 5 folds, greater than 10 folds, or greater than 100 folds in the amount of bound antibody or other linker (per unit time) to a polypeptide compared to a non-target polypeptide. A variety of immunoassay formats are suitable for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay conditions and formats that can be used to determine specific immunoreactivity.
[142] A specific nucleic acid sequence binding to the nucleic acid probe typically results in a greater than 2-fold increase, such as greater than 5 folds, greater than 10 folds, or greater than 100 folds in the amount of nucleic acid probe linked to a target nucleic acid sequence, compared to a non-target nucleic acid. A variety of ISH conditions are appropriate for selecting nucleic acid probes that specifically bind to a particular nucleic acid sequence.
[143] Specific binding portion or specific binding agent: A member of a specific binding pair. Specific binding pairs are pairs of molecules that are characterized by the fact that they are linked between i and the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 103 M '1 greater, 104 M'1 greater or 105 M'1 greater than the binding constant for both members of the binding pair with other molecules in a biological sample). Particular examples of specific binding portions include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidins, and protein A), nucleic acid sequences, and proteins / nucleic acids. Specifically bound portions may further include molecules (or portions thereof) that are specifically bound by such specific binding proteins.
[144] Substrate: A molecule acted by a catalyst, such as an enzyme. In one example, a substrate is 4-Chloro-1-naphthol (4-CN), or diaminobenzidine (DAB).
[145] Tissue: A collection of interconnected cells that perform a similar function in an organism.
[146] Tyramine: A compound that has the formula CsHuNO, also known as 4- (2-aminoethyl) phenol.
[147] Tiramide: A tyramine derivative, in which the amine functional group of a tyramine molecule formed an amide bond with a carbonyl-containing functional group. 111. Overview of Various Achievements A. Compositions
[148] Aspects of this disclosure refer to compositions that enhance the deposition of detectable portions in tissue samples, for example, sections of tissue such as those tested for the presence of markers, such as disease markers. Thus, compositions for enhancing the deposition of detectable portions in tissue samples, for example tissue sections, are disclosed in this document. An enhanced deposition provides an enhanced ability to easily detect and identify targets in a tissue sample, for example, by improving the quality, quantity and / or signal / noise ratio of detectable portions in tissue samples. In certain embodiments, the disclosed compositions and methods provided in this document increase and / or enhance enzyme renewal by increasing apparent enzyme oxidation rates, thereby enhancing the enzyme's ability to react with components of the composition and increasing the deposition of detectable portions at the specific target site, such as the site of a target molecule in a sample. In such embodiments, the enzyme products are detectable portions that are deposited on tissue samples. Exemplary composition components are further detailed in the following sections. 1. Intensifiers
[149] Revealed achievements use intensifiers or enhancement solutions to enhance enzymatic activity towards deposition of detectable moieties, for example, in the case of HRP by increasing the apparent enzyme reaction kinetics and thereby increasing the rate deposition of the enzyme substrate, such as the chromogenic reaction product of DAB and HRP. The disclosed enhancers can be used in solutions that comprise other components of the composition, or they can comprise a separate solution, in which the solution is separately added to other components of the composition. The solution can be an aqueous solution, a water-miscible organic solution or any combination thereof. Exemplary organic solutions include, but are not limited to, glycols, such as propylene glycol, dimethyl sulfoxide, tetrahydrofuran, dimethylformamide and any combination thereof. 1. Pyridine and Pyrimidine analogues
[150] Particular embodiments of the disclosed compositions include as pyrimidine analog enhancers and / or pyridine analogs that have the following general formulas (Formula 1 and Formula 2):

[151] In reference to Formula 1, R1, R2, R3 and R4 can independently be aliphatic, aryl, halogen, a portion containing heteroatom, and hydrogen. In some instances, halogen is iodine, bromine, chlorine or fluorine. In some examples, the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR6R7 where R6 and R7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination of them. In some examples, R1 and / or R3 are linked to R2 to form a fused ring system. In some examples, with reference to Formula 2, R5 is a heteroatom-containing moiety, such as hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR6R7 where R6 and R7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination thereof. In reference to Formula 1, in some examples, "A" is a heteroatom (other than sulfur), a carbon atom or any combination thereof. In reference to Formula 2, n is 1 to 5, such as 1, 2, 3, 4 or 5, for example 1 to 2, 2 to 3, 3 to 4, 4 to 5, 1 to 3, 2 to 4, 3 to 5, 1 to 4, 2 to 5 or 1 to 5. In specific examples, A is a carbon atom and n is 1. Exemplary pyrimidine analogs for inclusion in the disclosed compositions are pyrimidine and 2-hydroxypyrimidine and exemplary pyridine analogs are pyridine and 2-hydroxypyridine.
[152] In some examples, intensifiers are present in a solution, for example, to facilitate application from automated machines. In some examples, enhancers are present in the solution at a concentration of about 0.01 mM to about 100 mM, such as about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.10 mM, about 0.20 mM, about 0.50 mM, about 1.0 mM, about 2.0 mM, about 3.0 mM, about 5.0 mM, about 10.0 mM, about 20.0 mM, about 30.0 mM, about 40.0 mM, about 50.0 mM, about 75.0 mM, or about 100.0 mM, such as about 0.01 mM to about 0.10 mM, about 0.05 mM to about 0.50 mM, about 0.4 mM to about 1.0 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 10.0 mM, about 5.0 mM to about 50.0 mM, and about 20.0 mM to about 100.0 mM. Optional intensifiers
[153] In particular embodiments, the pyridine and pyrimidine analogue enhancers are used in conjunction with additional optional enhancers. In some embodiments, optional intensifiers are included in the same solution as the pyridine and pyrimidine analogue intensifiers and thus can be brought into contact with a tissue sample as a single composition, such as a single solution. In some cases, it may be desirable to include the optional intensifiers in a separate solution, for example, to avoid reactivity and / or reduced solubility. Thus, in some embodiments, the optional intensifiers are included in a different solution than the pyridine and pyrimidine analog intensifiers and thus can be brought into contact with a tissue sample from a separate solution, for example, as simultaneous delivery or staged delivery.
[154] Particular embodiments of the disclosed compositions contain optional enhancers that may or may not be used in combination with the pyrimidine analog and / or the pyridine analog, depending on such factors as the particular application or detectable moieties or enzymes, among others. In particular embodiments, a disclosed composition contains, as optional enhancers, heteroaryl enhancers that have the following general formula (Formula 3):
Formula 3
[155] In reference to Formula 3, R8, R9, R19θ Rn independently are aliphatic, aryl, halogen, a portion containing heteroatom, hydrogen or any combination thereof. In certain examples, halogen is iodine, bromine, chlorine or fluorine. In certain examples, the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR6R7 where R6 and R7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination thereof. In reference to Formula 3, "A" is a heteroatom (other than sulfur), a carbon atom or any combination thereof. In reference to Formula 3, "B" is oxygen, carbon, or nitrogen. In specific examples, A is a carbon atom and B is a nitrogen atom. In certain embodiments, heteroaryl enhancers for use in the disclosed compositions are selected from imidazole, L-histidine, thiazole, oxazole or any combination thereof. In some examples, intensifiers are present in a solution, for example, to facilitate application from automated machines.
[156] In some embodiments, the disclosed compositions contain, as optional enhancers, heteroaryl enhancers that have the following general formulas (Formula 4 and Formula 5):
Formula 4
Formula 5
[157] In reference to Formulas 4 and 5, R12 to R23 independently are aliphatic, aryl, halogen, a portion containing heteroatom, hydrogen or any combination thereof. In some instances, the halogen portion is iodine, bromine, chlorine or fluorine. In some examples, the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR6R7 where R6 and R7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination of them. In certain embodiments, R22 is a kind of radical, such as [O], or a negatively charged species, such as [O] ’. In reference to Formula 5, in particular embodiments, R21 and R17 comprise a gemmethyl di-methyl group. In reference to Formula 5 and / or 6, "A" is a heteroatom (other than sulfur), a carbon atom or any combination thereof. In specific examples, A is a nitrogen atom, an oxygen atom, a carbon atom or any combination thereof. In specific embodiments, heteroaryl enhancers include, but are not limited to, pyrimidine / V-oxide, pyridine A-oxide, / V-methyl morpholine (NMO), and 2,2,6,6-tetramethylpiperidine-l -oxy (TIME).
[158] In some embodiments, the disclosed compositions contain, as optional enhancers, non-organic and organic boronic acids. In some embodiments, organic boronic acids have the following general formula (Formula 6):
Formula 6
[159] In reference to Formula 6, R24, R25 and R26 are independently aliphatic, aryl, heteroaliphatic, heteroaryl or any combination thereof. In specific examples, two or more, such as 2 or 3, among R24, R25 and R26 are hydroxyl, with the remaining R24, R25 or R26 being aliphatic, aryl, heteroaliphatic or heteroaryl. In particular embodiments, R24, R25 and R26 independently are alkyl, alkenyl, alkynyl, and phenyl. In certain embodiments of the disclosed composition, organic boronic acids are boric acid, phenyl boronic acid, 4-AcHN-phenyl boronic acid or any combination thereof.
[160] In some embodiments, the disclosed compositions include phenolic compounds as optional enhancers. In some embodiments, phenolic compounds have the following general formula (Formula 7):
Formula 7
[161] In reference to Formula 7, R27, R28, R29, R30 and R31 are independently aliphatic, aryl, a portion containing heteroatom, halogen, hydrogen, or any combination thereof. In some instances, the halogen portion is iodine, bromine, chlorine or fluorine. In some instances, the heteroatom-containing moiety is hydroxyl, ether, silyl ether, ester, carboxylic acid, silyl, phosphonate, phosphine, amide, or NR6R7 where R6 and R7 independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl or any combination thereof. In particular embodiments, any two adjacent groups selected from R27, R28, R29, R30 and R31 can be linked to form an aromatic, non-aromatic or fused ring system. In additional particular embodiments, at least one of R27, R28, R29 R30 and R31 is selected from hydroxyl. Particular work achievements concern phenolic compounds as optional enhancers, such as pyrocatechol. [2] Detectable Portions
[162] The disclosed compositions allow for increased detection of detectable portions. These detectable portions can be selected from any portion that is capable of being used with tissue samples. Particular embodiments employ detectable portions selected from chromogens, fluorophores, tiramide conjugates, which are formed between tyramine and haptens, nanoparticles, fluorophores, and proteins or any combination thereof.
[163] Particular achievements use chromogens as detectable portions. Chromogens can be selected from any compound capable of producing a color change detectable by deposition on the tissue, for example, a tissue sample such as a section of tissue typically employed for pathological examination. In some examples, the detectable portion is deposited on sample tissue after being acted upon by an enzyme. As an example, the enzyme is directed to a target molecule in a tissue sample, the enzyme acts on the detectable portion, which in turn is deposited in the sample in the immediate vicinity of the enzyme, thus allowing the detection, quantification and / or location of the target molecule in a tissue sample. The detectable portions can be used in solutions that comprise other components of the composition, or they can comprise a separate solution, in which the solution is added separately to other components of the composition.
[164] Portions of specific binding can be designed to be directly attached to a label. Used in this way, the complete label / specific link (ie the probe) is placed in contact with the sample and the target is detected.
[165] Portions of specific binding may also be indirectly associated with a tag. In some examples, a first specific binding portion is contacted with a sample. The specific binding moieties can be based on nucleic acid or protein. The specific binding portion can be conjugated to another portion which is then attached, for example, by a secondary antibody or a non-peptide based binding portion, such as biotin. The secondary antibody or non-peptide binding pair can then be attached to a tag, such as an enzyme. In another example, a specific binding portion can be indirectly associated with a tag by conjugating the specific binding portion, directly or indirectly, to a peptide that has enzymatic activity. The enzyme activity is chosen so that by adding a substrate (s), the substrate (s) is / are converted into a detectable portion, or become a more active label.
[166] Exemplary non-limiting examples of enzyme / substrate pairs include the following: HRP / DAB with a chromogenic substrate or flurogenic substrate. Numerous other enzyme / substrate combinations are known to those skilled in the art. For a general review of these, please refer to Patents No. 22U.S. 4,275,149 and 4,318,980. When a probe is made from the indirect association of one or more additional molecules, the additional molecules can be called probe components.
[167] In some instances, the tag is indirectly conjugated to an antibody. For example, an antibody can be conjugated to biotin where the biotin selectively binds to avidin for subsequent detection. Alternatively, an antibody is conjugated to a small hapten and a tag is conjugated to an anti-hapten antibody. Thus, indirect conjugation of the label with the target portion can be achieved.
[168] When the probe includes an enzyme that reacts with a substrate to generate the detection tag, the substrate may be a chromogenic compound. There are numerous examples of such substrates. For example, many of such compounds can be purchased from Invitrogen, Eugene OR. Particular non-limiting examples of chromogenic compounds include nitrophenyl-β-D-galactopyranoside (ONPG), 5-bromo-4-chloro-3-iπdolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (Mil- Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-iπdolyl- β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), 10-acetyl-3,7-dihydroxyphenoxazione (ADHP), diamiπobeπzidiπa (DAB), tetramethylbenzidine (TMB), 2,2'-azino-di- [3-ethylbeπzotiazoliπa] (ABTS), o-dianisidine, 4-chlorone sulfonate (4-CN) (used in conjunction with DMPDA / DEPDA / MBTH / ADET, according to Kidwell, et al. Anal. Biochem., (1991), 192, 207), and o-phenylenediamine (OPD).
[169] Particular achievements use fluorophores as detectable portions. Fluorophores can be selected from compounds that exhibit fluorescence, including, but not limited to, fluoresceins, luminophores, coumarins, BODIPY dyes, resorufins and rodamines. Examples of particular fluorophores that can be used in the probes disclosed in this document are provided in Patent No. 2 U.S. 5,866,366 to Nazarenko et al., Such as 4-acetamido-4'-isothiocyanato-stilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1 acid -sulfonic (EDANS), 4-amino-N- [3-vinylsulfonyl) phenyl] naphthalim ida-3,5 (Lucifer Yellow VS), N- (4-anilino-1-naphthyl) maleimide, anthranilamide, Bright Yellow , coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcularine (Cumarana 151); cyanosine; 4 ', 6-diaminidino-2-phenylindole (DAPI); 5 ', 5 "-dibromopyrogalol-sulfonaphthalene (Bromopyrogalol Red); 7-diethylamino-3- (4'-isothiocyanatophenyl) -4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stylbene-2,2' -disulfonic; 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid; 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dansyl chlorine); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosine and derivatives such as erythrosine B and erythrosine isothiociaπate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiociaπate (FITC), and QFITC (XRITC); fluorescamine; IR144; IR1446; isothiociaπate; Malachite; 4-methylumbelliferone; ortho-cresolftaleiπa; nitrotirosiπea; pararosanilina; Feπol red; B-phycoerythrin; o-phthalialdehyde; pyrene ed derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (CIBACRON ™ Bright Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chlorine, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiociaπate, sulforodamine B , sulforodamine 101 and sulforodamine 101 sulfonyl chlorine derivative (Texas Red); N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiociaπate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; LuzCycler Red 640; Cy5.5; and Cy5 (many additional examples of fluorescent molecules can be found in The Handbook - A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR).
[170] In other embodiments, the detectable moiety may comprise a tiramide conjugate comprising a tyramine compound conjugated to a detectable moiety selected from nanoparticles, fluorophores and proteins. Certain embodiments use tyramide conjugates that comprise tyramine and a hapten in which the hapten is selected from oxazole, pyrazole, thiazole, benzofurazane, triterpene, urea, thiourea, nitroaryl, rotenoid, coumarin, cyclolignan, heterobiaryl, azoaryl, combinazepine of the same.
[171] Other embodiments contemplated by the present disclosure include tiramide conjugates that include a tyramine compound attached to a nanoparticle, such as a quantum dot. Other particular embodiments include a tyramine compound attached to a fluorophore, which can be selected from fluoresceins, luminophores, coumarins, BODIPY dyes, resorufins and rodamines. Still other particular embodiments concern a tyramine compound bound to a protein, which can be selected from an enzyme, such as horseradish peroxidase, glucose oxidase, β-galactosidase, β-glucuronidase or β-lactamase.
[172] In particular embodiments, the detectable tag or hapten is attached to the tyramine compound via a linker, such as an aliphatic linker, a heteroaliphatic linker or any other flexible attachment portion with comparable reactivities. For example, a tyramine compound can be covalently modified with a detectable tag through a heterobifunctional polyalkylene glycol linker such as a heterobifunctional polyethylene glycol (PEG) linker.
[173] A class of binders suitable for forming revealed tyramine-detectable conjugates and tyramide / hapten conjugates are aliphatic compounds, such as aliphatic hydrocarbon chains that have one or more unsaturation sites, or alkyl chains. The aliphatic chain typically also includes terminal functional groups, which include, by way of example and without limitation, a carbonyl-reactive group, amine-reactive group, a thiol-reactive group or a photo-reactive group, which facilitate coupling to detectable moieties and other desired compounds. The length of the chain can vary, but it typically has an upper practical limit of about 30 atoms. Chain links larger than about 30 carbon atoms have been shown to be less effective than compounds that have smaller chain links. Thus, aliphatic chain linkers typically have a chain length of about 1 carbon atom to about 30 carbon atoms. However, a person of ordinary skill in the art will note that if a particular ligand has more than 30 atoms, and still operates efficiently to link the detectable moiety to a tyramine compound, and the conjugate still works as desired, then such links chain-links are still covered by the scope of the present invention. Typical concentrations for tiramide / hapten conjugates comprising the disclosed ligands range from about 500 pM to about 100 pM. Even typical concentrations range from 5 pM to about 55 pM.
[174] Another class of useful binders are alkylene oxides. Alkylene oxides are represented in this document with reference to glycols, such as ethylene glycols. A person of ordinary skill in the art will observe that, as the number of oxygen atoms increases, the hydrophilic capacity of the compound may also increase. Thus, binders of the present disclosure typically have a formula of (-OCH2CH2O-) n where n is about 2 to about 15, but more particularly it is about 2 to about 8. In some examples, the detectable moieties are present in the solution in a concentration of about 0.01 mM to about 10.0 mM, such as about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.10 mM, about 0.20 mM, about 0.50 mM, about 1.0 mM, about 2.0 mM, about 3.0 mM, about 5.0 mM, or about 10.0 mM, such as about 0.01 mM to about 0.10 mM, about 0.05 mM to about 0.50 mM, about 0.4 mM to about 1.0 mM, about 0, 5 mM to about 2.0 mM, and about 1.0 mM to about 10.0 mM. [3] Specific Binding Portion Conjugates
[175] Particular embodiments concern the use of conjugates that comprise a specific binding moiety and an enzyme, in which the specific binding moiety is able to recognize and bind to a particular target in a sample. Binding specific portions can be selected from oligonucleotides, nucleic acids, proteins and peptides. Particular achievements concern the use of proteins, such as antibodies, as specific binding moieties.
[176] In particular embodiments, the specific binding moiety is linked to an enzyme. Examples of enzymes contemplated in the method can include oxidoreductases, such as peroxidases. Particular achievements concern the use of horseradish peroxidase, glutathione peroxidase or any other peroxidase that contains a heme portion.
[177] In certain embodiments, conjugates that comprise a specific binding moiety and a hapten can be used in conjunction with a second conjugate that comprises an anti-hapten antibody and an enzyme. In these particular embodiments, the specific binding portion, typically an antibody, can recognize a target in a sample and bind to it. The specific binding moiety is linked to a hapten, which will be recognized by the second conjugate which comprises the anti-hapten antibody and an enzyme. This second antibody conjugate will bind to the first conjugate, thereby binding the enzyme to the target. The hapten can be selected from oxazole, pyrazole, thiazole, benzofurazane, triterpene, urea, thiourea, nitroaryl, rotenoid, coumarin, cyclolignan, heterobiaryl, azoaryl, benzodiazepine or combinations thereof. Exemplary antibodies include, but are not limited to, rabbit IgG, mouse IgG, mouse IgM and goat IgG. Exemplary enzymes are as previously disclosed. In some examples, the specific binding portion and an enzyme conjugate are present in the solution at a concentration of about 0.01 mM to about 10.0 mM, such as about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.10 mM, about 0.20 mM, about 0.50 mM, about 1.0 mM, about 2.0 mM, about 3.0 mM , about 5.0 mM, or about 10.0 mM, such as about 0.01 to about 0.10, about 0.05 to about 0.50, about 0.4 to about 1.0, about 0.5 to about 2.0, and about 1.0 to about 10.0. [4] Other Components
[178] Particular embodiments relate to compositions that additionally comprise Group I or Group II salt containing metals that have an MXn or MX formula, where M is a Group I or Group II metal, and X is selected from halide, such as fluorine, bromine, chlorine and iodine; and ions that contain oxygen, such as carbonate, hydroxide and phosphate. Particular achievements concern the use of Group I metal selected from sodium, lithium, cesium and potassium. Other particular achievements concern the use of Group II metals selected from magnesium, calcium, strontium and barium. Particular embodiments use magnesium and / or calcium salt enhancers selected from calcium chlorine, magnesium chlorine and calcium carbonate. In some examples, optional enhancers are present in the solution at a concentration of about 0.01 mM to about 100 mM, such as about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.10 mM, about 0.20 mM, about 0.50 mM, about 1.0 mM, about 2.0 mM, about 3.0 mM, about 5.0 mM, about of 10.0 mM, about 20.0 mM, about 30.0 mM, about 40.0 mM, about 50.0 mM, about 75.0 mM, or about 100.0 mM, as such as about 0.01 mM to about 0.10 mM, about 0.05 mM to about 0.50 mM, about 0.4 mM to about 1.0 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 10.0 mM, about 5.0 mM to about 50.0 mM, and about 20.0 mM to about 100.0 mM.
[179] Particular embodiments relate to compositions that further comprise oxidants, inhibitors and surfactants as components that can be used in any combination with any of the components previously disclosed. Oxidizers can include any compound capable of effectively activating the enzyme. Particular achievements concern the use of peroxides, such as hydrogen peroxide, as oxidants used to activate the enzyme. Typically, the oxidant is 0.03% hydrogen peroxide.
[180] An inhibitor can be selected from any compound capable of effectively inhibiting the enzyme after it has reacted sufficiently in a way that results in the deposition of the detectable portion. Particular achievements concern inhibitors selected from peroxidases. Typically, hydrogen peroxide is used as an inhibitor. Certain achievements concern the use of 3% hydrogen peroxide as an inhibitor. In particular embodiments, the inhibitor is added to the sample after the addition of the other components of the composition.
[181] Particular embodiments of this disclosure relate to compositions for use in detecting a target molecule in a sample, such as a tissue sample. In some embodiments, commercially viable compositions comprise a pyrimidine analog and / or a pyridine analog, an optional enhancer, a detectable moiety, a specific binding moiety conjugate, an enzyme, an oxidant and a surfactant. These components of the composition can be added in any order and any combination that results in the effective deposition of the detectable portion on the target in the sample. The compositions can be used in conjunction with a second antibody, which is conjugated to an enzyme, an oxidizer and a dye.
[182] In particular work embodiments, the pyrimidine analog and / or the pyridine analog is selected from 2-hydroxypyrimidine and / or 2-hydroxypyridine. In particular work embodiments, the specific binding portion conjugate is a haptenated IgG antibody conjugate and the secondary antibody comprises an anti-hapten multimer HRP conjugate.
[183] Particular achievements revealed relate to the use of a surfactant. Surfactants are classified as anionic, cationic or non-ionic, depending on their mode of chemical action. Non-ionic surfactants work through a hydrogen bonding mechanism. In addition, surfactants reduce the interfacial tension between two liquids. A surfactant molecule typically has an ionic or polar "head" and a non-polar hydrocarbon "tail." By dissolving in water, the surfactant molecules aggregate and form micelles, in which the non-polar tails are oriented inward and the ionic or polar heads are oriented outwards towards the aqueous environment, the non-polar tails create a non-polar "pocket" in the micelle. Non-polar compounds in the solution are sequestered in the pockets formed by the surfactant molecules, thus allowing compounds non-polar remain mixed in the aqueous solution In particular revealed embodiments, the surfactant can be used to produce uniform spreading of reagents across a section of tissue as well as to decrease background staining.
[184] Examples of surfactants include, but are not limited to, polyoxyethylene alkyl ether, where alkyl is (CH2) M and oxyethylene is (C2H4O) N, where M is an integer from 5 to 16, from 8 at 14 or from 10 to 12 and N is an integer from 10 to 40, from 15 to 30, or from 20 to 28. In one embodiment, the surfactant is polyoxyethylene lauryl ether which has a formula (C2H4O) 23Ci2H2sOH. In another embodiment, the surfactant is a polyoxyethylene (20) sorbitan monoalkylate, wherein the monoalkylate comprising between 8 and 14 carbons. In another embodiment, the surfactant is a linear secondary alcohol polyoxyethylene that has a formula Ci2-i4H25-2θO (CH2CH2θ] x, where x is equal to an integer between 2 and 12. In yet another embodiment, the surfactant is a polyoxyethylene octyl phenyl ether Exemplifying surfactants are marketed under the name: Brij® 35, TWEEN®, Tergitol ™, Triton ™, Ecosurf ™, Dowfax ™, polysorbate 80 ™, BigCHAP, Deoxy BigCHAP, IGEPAL®, Saponin, Thesit®, Nonidetet ®, Pluronic F- 68, digitonin, deoxycholate and the like .. Particular work performed presented concerns the use of surfactants selected from Brij® 35, TWEEN®, Tergitol ™, Triton ™. 8. Method of Detecting a Target Molecule
[185] The compositions shown are particularly useful for the detection of target molecules in samples because they act synergistically to provide maximized deposition during IHC or ISH tissue staining. Therefore, the present description provides a method of detecting a target molecule in a sample, such as a tissue sample. In some embodiments, the method includes contacting a sample with an intensifying solution that includes a pyrimidine analogue and / or a pyridine analogue, as described in the previous section (Section A), placing the sample in contact with an enzyme and put the sample in contact with a detectable portion capable of being detected using deposition or fluorescent techniques. In some examples, the enzyme acts on a substrate to catalyze the production of the detectable portion, which is deposited on the sample at the site of the target molecule, thus allowing the detection of the target molecule. The detectable portion is detected, thereby detecting the target molecule in a sample. In some examples, the intensity and / or location of a signal produced by the detectable portion is measured, for example, to determine the quantity and / or the location of the target molecule in the tissue sample. The target can be any molecule of interest for which the presence, location and / or concentration must be determined. Examples of molecules of interest include proteins and nucleic acid sequences. In some embodiments, the pyrimidine analog is pyrimidine and / or 2-hydroxypyrimidine. In some embodiments, the pyridine analog is 2-hydroxypyridine.
[186] In some embodiments, the enzyme is immobilized on the target by incubating the sample with an enzyme conjugate that binds to the target. The enzyme can be conjugated to any portion capable of binding the target, for example, conjugated to an antibody or nucleic acid that specifically recognizes the target molecule. Suitable portions include, but are not limited to, antibodies, nucleotides, oligonucleotides, proteins, peptides or amino acids.
[187] In other embodiments, immobilizing the enzyme is a multi-step process. For example, the sample can be incubated with a first portion (for example, an antibody, nucleotide, oligonucleotide, protein, oligopeptide, peptide or amino acid) that binds to the target. The sample can then be incubated with an enzyme conjugate that comprises a portion that is capable of binding to the first portion. In some embodiments, where the first portion is an antibody to the target, the two-step process can be more versatile because it allows the user to employ an “universal” enzyme-antibody conjugate. For example, if the first antibody is a monoclonal rabbit antibody, the enzyme-antibody conjugate can include an antibody that is capable of binding to any monoclonal rabbit antibody, for example, a secondary antibody. The multi-step process can eliminate the need to generate an enzyme-antibody conjugate that is suitable for each target.
[188] In some embodiments, the first portion may be a labeled probe, such as a labeled oligonucleotide. After the probe is hybridized to the sample, a first antibody that recognizes the tag is introduced and binds to the labeled probe. The first antibody can be an enzyme-antibody conjugate. However, if the first antibody is not conjugated to an enzyme, an enzyme-antibody conjugate is introduced in which the antibody portion of the conjugate recognizes and binds to the first antibody.
[189] In some embodiments, the enzyme is a peroxidase, such as horseradish peroxidase or glutathione peroxidase or an oxidoreductase. Therefore, suitable conditions are selected for the enzyme reaction, such as a salt concentration and pH that allows the enzyme to perform its desired function, for example, converting the substrate into a detectable portion that is deposited in the tissue sample at the target molecule site. . The reaction is carried out at a temperature that is suitable for the enzyme. For example, if the enzyme is horseradish peroxidase, the reaction can be carried out at about 35 to 40 ° C.
[190] In some instances, a detectable moiety is a chromogen, a fluorophor, a hapten or a protein. In specific examples, the detectable moiety is 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole or tetramethylbenzidine or a reaction product thereof. Additional examples of chromogens for use in the methods described are given in the previous section (Section A).
[191] In other examples, a detectable portion is a fluorophor, such as a fluorescein, a luminophore, a coumarin, a BODIPY dye, a resorufin or a rhodamine. Additional examples of fluorophores for use in carrying out the presented method are given in the previous section (Section A).
[192] In other examples, a detectable moiety is a hapten, such as an oxazole, a pyrazole, a thiazole, a benzofurazane, a triterpene, a urea, a thiourea, a nitroaryl, a rotenoid, a coumarin, a cyclolignan, a heterobiaril , an azoaryl or benzodiazepine.
[193] In specific embodiments, the detectable moiety is conjugated to tyramine, for example, through a linker, such as an aliphatic or heteroaliphatic linker of about 1 to about 30 carbon atoms in a chain. In specific embodiments, the binder is an alkylene oxide, such as ethylene glycol or a polymer thereof, for example, a polymer with 1 to about 15 ethylene glycol units. When tyramines are used with the described methods, amplification of the output signal can be used to further amplify the generated signal. Amplification of the tiramide signal uses the catalytic activity of a peroxidase enzyme to covalently link a tyramine or tyramine-derived residue to a solid phase. The solid phase can be, for example, protein components of cells or cellular structures that are immobilized on a substrate, such as a microscope slide. Some peroxidase enzymes (for example, horseradish peroxidase), in the presence of a peroxide, can catalyze the dimerization of phenolic compounds. Therefore, if tyramine is added to a sample that contains protein in the presence of horseradish peroxidase and peroxide (for example, hydrogen peroxide), the phenol tyramine group can form a dimer with the phenol group of a tyrosine amino acid.
[194] Only tyramine molecules in close proximity to the immobilized enzyme will react and form dimers with tyrosine residues in or around the immobilized enzyme, including tyrosine residues in the enzyme itself, tyrosine residues in the antibody to which the enzyme is conjugated and / or tyrosine residues in the sample that are close to the immobilized enzyme, such as about 100 nm, about 50 nm, about 10 nm or about 5 nm of the immobilized enzyme. For example, the tyrosine residue can be at a distance of about 10 angstroms to about 100 nm, about 10 angstroms to about 50 nm, about 10 angstroms to about 10 nm, or about 10 angstroms to about 5 nm of the immobilized enzyme. Such close linkage allows the target to be detected with at least the same degree of specificity as conventional staining methods used with IHC and / or ISH. For example, embodiments of the presented method allow subcellular structures to be distinguished, for example, nuclear membrane versus the nuclear region, cell membrane versus the cytoplasmic region, etc.
[195] Once the enzyme is immobilized in the sample, the tiramide conjugate is introduced under suitable conditions to allow the enzyme to react with the tiramide. Typically, the enzyme is a peroxidase, like horseradish peroxidase. Under such conditions, the tiramide reacts with the peroxide and the enzyme, converting the tiramide into an active form that covalently binds to the sample, typically by binding a tyrosine residue in the vicinity of the immobilized enzyme, including tyrosine residues in the immobilized enzyme itself . After the tiramide conjugate is attached to the sample, its presence is detected by suitable means, for example, by virtue of a detectable portion attached to the tiramide.
[196] In some embodiments, the sample is additionally brought into contact with at least one optional intensifier, such as one or more of a heteroaryl compound, a salt containing Group I or Group II metal, a compound containing boron, a phenol compound. Optional exemplifying intensifiers are described in the previous section (Section A). In some embodiments, the sample is additionally brought into contact with an oxidizer, such as a peroxidase, for example, hydrogen peroxidase. In some embodiments, the sample is additionally placed in contact with a surfactant, such as Brij® 35, TWEEN®, Tergitol ™ and Triton ™. In some embodiments, the sample is additionally brought into contact with an antioxidant, such as sodium stannate, sodium metabisulfate and sodium bisulfate.
[197] The realizations of the method, as shown in this document, can be performed manually or automatically, for example, in an automated tissue processing instrument. Automated systems are typically, at least partially, if not substantially entirely, under computer control. Because automated systems are typically at least partially computer-controlled, certain embodiments of the present description also refer to one or more tangible computer-readable means that store computer-executable instructions for getting a computer to perform the presented achievements of the method. C. Samples and targets
[198] Samples include biological components and are, in general, suspected of including one or more target molecules of interest. Target molecules can be on the surface of cells and cells can be in suspension or in a section of tissue. Target molecules can also be intracellular and detected by cell lysis or penetration of the cell by a probe. A person of ordinary skill in the art will find that the method for detecting target molecules in a sample will vary depending on the type of sample and probe being used. Sample collection and preparation methods are known in the art.
[199] Samples for use in carrying out the method and with the composition presented here, such as a tissue or other biological sample, can be prepared using any method known in the art by an individual of skill in the art. Samples can be obtained from an individual for routine screening or from an individual who is suspected of having a disorder, such as a genetic abnormality, infection or neoplasm. The described achievements of the presented method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., called “normal” samples. Such normal samples are useful, among other things, as controls for comparison with other samples. Samples can be analyzed for many different purposes. For example, the samples can be used in a scientific study or for the diagnosis of a suspected disease, or as prognostic indicators for treatment success, survival, etc.
[200] Samples can include multiple targets that can be specifically linked by a reporter probe or molecule. The targets can be sequences of nucleic acid or proteins. Through this description, when reference is made to a target protein, it is understood that the nucleic acid sequences associated with that protein can also be used as a target. In some instances, the target is a protein or nucleic acid molecule from a pathogen, such as a virus, bacteria or intracellular parasite, such as from a viral genome. For example, a target protein can be produced from a target nucleic acid sequence associated with (for example, correlated with, casually implicated in, etc.) a disease.
[201] A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example, a target nucleic acid sequence can have at least about 10 nucleic acid residues or at least about 20, 30, 50, 100, 150, 500, 1,000 residues. Similarly, a target polypeptide can vary substantially in size. Without limitation, the target polypeptide will include at least one epitope that binds to a specific peptide antibody, or fragment thereof. In some embodiments in which the polypeptide may include at least two epitopes that bind to a specific peptide antibody, or fragment thereof.
[202] In specific, non-limiting examples, a target protein is produced by a target nucleic acid sequence (eg, genomic target nucleic acid sequence) associated with a neoplasm (eg, a cancer). Several chromosomal abnormalities (including translocations and other reorganizations, amplification or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of the target molecule is produced by an amplified or excluded nucleic acid sequence (e.g., genomic target nucleic acid sequence) in at least a subset of cells in a sample.
[203] Oncogenes are known to be responsible for several human malignancies. For example, chromosomal reorganizations that involve the SYT gene located in the 18q11.2 chromosome disruption region are common among synovial sarcoma soft tissue tumors. The translocation of t (18q11.2) can be identified, for example, using probes with different tags: the first probe includes FPC nucleic acid molecules generated from a target nucleic acid sequence that extends distally from the SYT gene and the second probe includes FPC nucleic acid generated from a target nucleic acid sequence that extends over 3 'or in the vicinity of the SYT gene. When the probes corresponding to these target nucleic acid sequences (for example, genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells, which do not have a t (18q11.2) in the gene region SYT, display two fusion signals (generated by the two tags in close proximity), reflecting the two intact copies of SYT. Abnormal cells with a t (18q11.2) exhibit a single fusion signal.
[204] In other examples, a target protein produced from a nucleic acid sequence (eg, genomic target nucleic acid sequence) is selected, which is a tumor suppressor gene that is deleted (lost) in malignant cells . For example, the p16 region (including D9S1749, D9S1747, p16 (INK4A), p14 (ARF), D9S1748, p15 (INK4B) and D9S1752) located on chromosome 9p21 is excluded in certain bladder cancers. Chromosomal exclusions involving the distal region of the short arm of chromosome 1 (covering, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1 and SHGC-1322), and the pericentromeric region (for example, 19p13-19qq13) of chromosome 19 (which covers, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2 and GLTSCR1) are typical molecular characteristics of certain typical of solid tumors of the central nervous system.
[205] The aforementioned examples are provided for illustrative purposes only and are not intended to be limiting. Several other cytogenetic anomalies that are correlated to neoplastic transformation and / or growth are known to those of ordinary skill in the art. Target proteins that are produced by nucleic acid sequences (for example, target genomic nucleic acid sequences), that have been correlated to neoplastic transformation and that are useful in the methods described, also include the EGFR gene (7p12; for example, GENBANK ™ Access NC_000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; for example, GENBANK ™ Access NC_000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipase lipoprotein gene ( LPL) (8p22; for example, GENBANK ™ Accession No. NC_000008, nucleotides 19841058-19869049), RB1 (13q14; for example, GENBANK ™ Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17p13.1; by example, GENBANK ™ Accession No. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; for example, GENBANK ™ Accession No. NC_000002, complement, nucleotides 151835231-151854620), CHOP (12q13; for example , GENBANK ™ Accession No. NC_000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; by example, GENBANK ™ Accession No. NC_000016, nucleotides 31098954-31110601), FKHR (13p14; for example, GENBANK ™ Accession No. NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; for example, GENBANK ™ Accession No. NC_000002, complement, nucleotides 29269144-29997936), heavy chain of lg, CCND1 (11q13; for example, GENBANK ™ Accession No. NC_000011, nucleotides 69165054.69178423), BCL2 (18q21.3; for example, GENBANK ™ Accession No. NC_000018, complement, nucleotides 58941559-59137593), BCL6 (3q27 ; for example, GENBANK ™ Accession No. NC_000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; for example, GENBANK ™ Accession No. NC_000001, complement, nucleotides 59019051-59022373), TOP2A (17q21 -q22; for example, GENBANK ™ Accession No. NC_000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; for example, GENBANK ™ Accession No. NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22 .3, for example, GENBANK ™ Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; for example, GENBANK ™ Accession No. NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; for example, GENBANK ™ Accession No. NC_000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; for example, GENBANK ™ Accession No. NC_000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; for example, GENBANK ™ Accession No. NC_000002, complement, nucleotides 222772851-222871944 ), PAX7 (1p36.2-p36.12; for example, GENBANK ™ Accession No. NC_000001, nucleotides 18830087-18935219), PTEN (10q23.3; for example, GENBANK ™ Accession No. NC_000010, nucleotides 89613175-89716382 ), AKT2 (19q13.1 -q13.2; for example, GENBANK ™ Accession No. NC_000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; for example, GENBANK ™ Accession No. NC_000001, complement, nucleotides 40133685-40140274), REL (2p13-p12; for example, GENBANK ™ Accession No. NC_000002, nucleotides 60962256-61003682) and CSF1R (5q33-q35; for example, GENBANK ™ Accession No. NC_000005, complement, nucleotides 149413051 -149473128).
[206] In other examples, the target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus or microorganism-derived nucleic acid sequence (eg, genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism. For example, the target peptide, polypeptide or protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparume other species Plasmodium, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria and Babesia species).
[207] In some examples, the target protein is produced from a nucleic acid sequence (eg, genomic target nucleic acid sequence) from a viral genome. Exemplifying viruses and corresponding genomic sequences (Seq Access No. Ref GENBANK ™ in parentheses) include human adenovirus A (NC_001460), human adenovirus B (NC_004001), human adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266), human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus (V01109; GI: 60851) human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human coronavirus HKU1 (NC_0077) NL63 (NC_005831), human coronavirus OC43 (NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrovirus V9 (human NC_004295), foamy virus (NC_001736), human herpesvirus 1 (herpes simplex virus type 1) (NC_001806), human herpesvirus 2 (herpes simplex virus type 2) (NC_001798), human herpesvirus 3 (Varicella zoster virus) (NC_001348), herpesvirus human 4 type 1 (Epstein-Barr virus type 1) (NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human herpesvirus 5 strain AD169 (NC_001347), human herpesvirus 5 strain Merlin Strain (NC_006273), human herpesvirus 6A (NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7 (NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P (NC_009333), human immunodeficiency virus 1 (NC_001802), virus human immunodeficiency 2 (NC_001722), human metapneumovirus (NC_004148), human papillomavirus-1 (NC_001356), human papillomavirus-18 (NC_001357), human papillomavirus-2 (NC_001352), human papillomavirus-54 (NC_001676), human papilomavirus (61_ 611676), NC_001694), human papillomavirus-cand90 (NC_004104), human papillomavirus RTRX7 (NC_004761), human papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC_008189), human papillomavirus type 103 (NC_008188), human papillomavirus (type 107), human papillomavirus human papillomavirus type 16 (N C_001526), human papillomavirus type 24 (NC_001683), human papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human papillomavirus type 34 (NC_001587), human papillomavirus type 4 (NC_001457), human papillomavirus13 type 41 (NC) , human papillomavirus type 48 (NC_001690), human papillomavirus type 49 (NC_001591), human papillomavirus type 5 (NC_001531), human papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC_001593), human papillomavirus type 60 (NC_001693), type 63 human (NC_001458), type 6b human papillomavirus (NC_001355), type 7 human papillomavirus (NC_001595), type 71 human papillomavirus (NC_002644), type 9 human papillomavirus (NC_001596), type 92 human papillomavirus (NC_004500), type human papillomavirus 96 (NC_005134), human parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC_003443), human parainfluenza virus 3 (NC_001796), human parecovirus (NC_001897), human parovirus 4 (NC_00701 8), human parovirus B19 (NC_000883), human respiratory syncytial virus (NC_001781), human rhinovirus A (NC_001617), human rhinovirus B (NC_001490), human spumaretrovirus (NC_001795), human T-lymphotropic virus 1 (NC_001436), T virus - human lymphotropic 2 (NC_001488).
[208] In certain examples, the target protein is produced from a nucleic acid sequence (eg, genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, for example, HPV16, HPV18). In other examples, the target protein produced from a nucleic acid sequence (for example, genomic target nucleic acid sequence) is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (for example, Hepatitis Virus C), a Coronavirus (for example, SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV) or a Herpes Simplex Virus (HSV). D. Sample Preparation
[209] The tissue samples described in this document can be prepared using any method currently known or developed in the future in the art. In general, tissue samples are prepared by fixing and immersing the tissue in a medium.
[210] In some instances, a means of integration is used. An integration medium is an inert material into which tissues and / or cells are integrated to help preserve them for future analysis. Integration also allows tissue samples to be sliced into thin sections. Integration media include, but are not limited to, paraffin, celloin, OCT ™ compound, agar, plastics or acrylics.
[211] Many means of integration are hydrophobic; therefore, inert material may need to be removed prior to histological or cytological analysis, which uses primarily hydrophilic reagents. The term dewaxing or wax removal is widely used in this document to refer to the complete or partial removal of any type of integration medium from a biological sample. For example, sections of tissue integrated with paraffin have the wax removed by passing through organic solvents, such as toluene, xylene, limonene or other suitable solvents.
[212] The process of fixing a sample may vary. Fixing a tissue sample preserves cells and tissue constituents in a state as close to life-like as possible and allows them to undergo preparatory procedures without significant changes. The fixation for the autolysis and bacterial decomposition processes that start with cell death and stabilizes the cellular and tissue constituents so that they support the subsequent stages of tissue processing, such as for IHC or ISH.
[213] Tissues can be fixed by any suitable process, including perfusion or by submersion in a fixative. Fixatives can be classified as crosslinking agents (such as aldehydes, for example, formaldehyde, paraformaldehyde and glutaraldehyde, as well as non-aldehyde crosslinking agents), oxidizing agents (for example, complex metal ions, such as osmium tetroxide and chromic acid ), protein denaturalizing agents (eg, acetic acid, methanol and ethanol), fixers of unknown mechanism (eg, mercuric chloride, acetone and picric acid), combination reagents (eg, Carnoy's fixative, metacam, fluid Bouin's, B5's fixative, Rossman's fluid and Gendre's fluid), microwaves and various fixers (for example, excluded volume fixation and steam fixation). Additives can also be included in the fixative, such as buffers, detergents, tannic acid, phenol, metal salts (such as zinc chloride, zinc sulphate and lithium salts) and lanthan.
[214] The fixative most commonly used in sample preparation for HCI is formaldehyde, generally in the form of a formalin solution (4% formaldehyde in a buffer solution, referred to as 10% buffered formalin). In one example, the fixative is 10% neutral buffered formalin. E. Counterstaining
[215] Counterstaining is a method of post-treating samples after they have already been stained with agents to detect one or more targets, so that their structures can be more readily viewed under a microscope. For example, a counterstain is optionally used previously to lamination to yield the most distinct immunohistochemical strain. Counterstains differ in color from a primary color. Several counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, giemsa, alcian blue and fast nuclear red.
[216] In some examples, more than one color can be mixed to produce the counter color. This provides flexibility and the ability to choose colorations. For example, a first coloring can be selected for the mixture that has a particular attribute, but does not yet have a different desired attribute. A second color can be added to the mixture that shows the absence of the desired attribute. For example, toluidine blue, DAPI and pontamine sky blue can be mixed together to form a counter color. F. Imaging
[217] Certain aspects, or all, of the presented achievements can be automated and facilitated by computer analysis and / or image analysis system. In some applications, accurate color ratios are measured. In some embodiments, light microscopy is used for image analysis. Certain achievements presented involve obtaining digital images. This can be accomplished by attaching a digital camera to a microscope. Digital images obtained from stained samples are analyzed using image analysis software. Color can be measured in several different ways. For example, color can be measured as values for red, blue and green; tone, saturation and intensity values; and / or by measuring a specific wavelength or wavelength range using a spectral imaging camera.
[218] One presented embodiment involves the use of bright field imaging with chromogenic dyes. While the light in the visible spectrum is transmitted through the dye. The dye absorbs light of certain wavelengths and transmits other wavelengths. This changes the light from white to colored, depending on the specific wavelengths of transmitted light.
[219] Samples can also be evaluated qualitatively and semi-quantitatively. The qualitative evaluation includes the evaluation of the staining intensity, identification of the positively stained cells and the intracellular compartments involved in the staining and the evaluation of the general sample or slip quality. Separate evaluations are carried out on the test samples and this analysis may include a comparison to average values to determine whether the samples represent an abnormal state. G. Kits
[220] The embodiments presented provide, in part, kits for carrying out various embodiments of the method of the invention. Examples of such kits include those useful for cholesterol analysis, pregnancy kits, cancer diagnosis kits, etc.
[221] In some embodiments, the kit includes a pyrimidine analog and / or a pyridine analog that has a formula, as described in Section A.
[222] In some embodiments, the kit includes an enzyme, such as an oxide reductase or a peroxidase, such as horseradish peroxidase or glutathione peroxidase. In some examples, the kit includes a specific binding portion, such as an antibody or nucleic acid that specifically binds to a target molecule. In some examples, the specific binding portion and the enzyme are linked to each other.
[223] In some embodiments, the kit includes a detectable portion capable of being detected using deposition or fluorescent techniques or an enzyme substrate that produces the detectable portion after reaction with the enzyme. In some instances, the detectable portion is a fluorophor (such as a fluorescein, a luminophor, a coumarin, a BODIPY dye, a resorufin or a rhodamine), a hapten (such as oxazole, pyrazole, thiazole, benzofurazane, triterpene, urea, thiourea, nitroaryl, rotenoid, coumarin, cyclolignan, heterobiaryl, azoaryl, benzodiazepine), a protein or chromogen (such as 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole or tetramethylbenzidine). The kit can optionally include at least one optional enhancer, such as a heteroaryl compound, a salt containing Group I or Group II metal, a compound containing boron and a phenol compound, for example, those described in Section A.
[224] In some embodiments, the kit includes an oxidizer, such as a peroxide, for example, hydrogen peroxide.
[225] In some embodiments, the kit includes a surfactant, such as Brij® 35, TWEEN®, Tergitol ™ and Triton ™.
[226] In some embodiments, the kit includes an antioxidant selected from sodium stannate, sodium metabisulfate and sodium bisulfate.
[227] In some embodiments, the kit includes a copper jaw. The kit may include additional components, including antibodies, labeled hapten probes, and other reagents needed to perform IHC and / or ISH by chromogenic detection. Such kits can be used, for example, by a clinician or physicist with an aid to select an appropriate therapy for a particular patient or for diagnostic purposes.
[228] Particular achievements refer to the use of kits that comprise an inhibitor, such as 3% H2O2; a Universal Multimer HRP, such as an anti-mouse / goat rabbit conjugated to HRP; a peroxide, such as 0.03% H2O2; a chromogen, like DAB; and a copper mordant. This kit is called ultraView ™, and can be used in combination with the described intensifiers. H. Automated Achievements
[229] An individual of ordinary skill in the art will find that the achievements of the method presented in the present invention for the chromogenic detection of two or more molecules can be automated. Ventana Medical Systems, Inc. is the assignee of several US patents that present systems and methods for performing automated analyzes, including US Patents No. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943 .029 and US Published Orders No. 20030211630 and 20040052685. Particular procedural accomplishments were conducted using various automated processes. IV. Work Examples
[230] The following examples are provided to illustrate certain specific characteristics of work achievements. The scope of the present invention is not limited to those characteristics exemplified by the following examples. EXAMPLE 1 IHC FABRIC COLORING WITH IMIDAZOLE
[231] IHC staining of bcl2 in tonsil was performed using DAB Universal ultraView ™ Chromogen enhanced with imidazole. Imidazole (10 mM -100 mM) significantly increased the intensity of DAB staining (see Figures 1 and 2). However, as the desired DAB signal increased, the observed background signal also increased. The highest concentrations of imidazole were shown to be incompatible, causing DAB to precipitate from the reformulated DAB Chromogen solution. EXAMPLE 2 KINETIC SCREENING OF INTENSIFIERS
[232] A plaque assay was developed to independently screen potential compounds and to better understand the observed enhanced DAB deposition, as demonstrated in Example 1. The additives were directly added to a well containing the necessary reagents of the ultraView ™ Detection Kit (VMSI 760-500: 253-4290, 253-4292 and 253-4293) in 1X Reaction Buffer (VMSI 950-300) and 0.1% fish gelatin. Fish gelatine was used to help disperse oxidized DAB and inhibit its precipitation from the solution. The ultraView ™ Detection Kit was diluted to measure the formation of oxidized DAB by UV-VIS at 455 nm. Additives were tested at set concentrations to determine the intensification of the DAB oxidation reaction. The data were plotted and the maximum apparent speed (Vmax) was calculated at each level of additive concentration.
[233] The initial test of 4-acetylamidophenyl boronic acid and imidazole demonstrated an increased reaction rate for HRP as the concentration of any of the additives increased (See Figures 3 and 4, respectively). The apparent Vmax for HRP oxidized DAB was 18.5 mOD / minute without any intensification. The addition of 10 mM of imidazole increased the apparent Vmax by 56% and 10 mM of 4-acetylamidophenyl boronic acid increased the apparent Vmax by 77%. (Percentage increase Vmax = [(Intensified Vmax - uView Vmax) / uView Vmax] x 100%).
[234] Intensified by the above results, other buffers that could be used as potential intensifiers have been examined. In addition to the conceptual use of imidazole analogues, oxidation reactions mediated by peroxide facilitated by L-histidine were explored. 50 mM L-histidine increased the apparent HRmax V by 138% (see Figure 5). As with a higher concentration of imidazole, 50 mM of L-histidine also caused the precipitation of DAB from the reformulated DAB Chromogen solution. A concentration of 10 mM L-histidine was used with reformulations of DAB. The apparent Vmax of HRP was increased by 18% with 10 mM L-histidine. Borate buffers were also tested. The buffering capacity of boric acid is not varying from the desired pH (pH range ranging from about 1 to about 7.9, final pH ranging from about 2 to about 3) for the formulation of DAB because acid boric acid has an effective pH buffer range of about 8.5 to about 10.2. The addition of 10 mM boric acid intensified the HRP apparent Vmax by 265%, however, at 50 mM, boric acid greatly intensified the HRP apparent Vmax by 592% (see Figure 6).
[235] Other heterocyclic compounds were examined to find a new class of intensifiers that would further increase the apparent rate of HRP. Pyrimidine analogs have been revealed to be a new class of enhancers. A summary of the test results for all HRP-mediated DAB oxidation enhancers can be found in Table 1. TABLE 1 INFLUENCE OF INTENSIFIERS ON THE APPARENT VMP FOR HRP OXIDED DAB WHEN ADDED TO THE ULTRA VIEW ™ DETECTION KlT.



[236] The optical density of oxidized DAB was monitored at 455 nm. (Sat) = Rate of reaction that was saturated by the beginning of the UV-VIS analysis. (Sol) = Solubility problem occurred at room temperature. The heat was required to dissolve the additive in the reaction buffer. (1) The additive was not soluble at room temperature.
[237] The addition of 10 mM pyrimidine greatly increased the apparent HRmax by 462%. This increase in the apparent rate of oxidation of DAB was higher than that observed with other heterocyclic intensifiers (nuclear structures of imidazole, thiazole and oxazole). Increasing the concentration of pyrimidine to 50 mM provided a modest increase in HRP Vmax over 10 mM (570%) (see Figure 7). A reformulation of the DAB chromogen with pyrimidine presents a potential problem due to the high vapor pressure (bp «124 ° C). Therefore, other pyrimidine analogs have been investigated in order to find a suitable alternative that does not have volatility problems.
[238] Pyrimidine nucleotide bases (thymine, uracil and cytosine) increased the apparent rate of HRP, however, they suffer from water solubility problems. Both 2-hydroxypyrimidine (see Figure 8) and pyrimidine-N-oxide have been found to provide increased or similar apparent rates over pyrimidine and have no solubility or volatility problems. Five- and six-membered heterocyclic N-oxides have previously been shown to increase the apparent rate of HRP-based oxidation of both oligo and polysaccharides, namely cellulose oxidation. DAB reformulations with pyrimidine-N-oxide lost functionality and stopped staining over time. However, pyrimidine-N-oxide is still useful in an intensification solution, if added to HRP-mediated oxidation reactions in tissue. In addition to pyrimidine, 10 mM 2-hydroxypyridine increased the apparent HRmax V (157%). Benzimidazole, methylene blue, phenothiazine and 4-dimethylaminopyridine did not provide any enhancement. EXAMPLE 3 SYNERGIC AND ANTAGONIC EFFECTS FOR INTENSIFIERS
[239] The Example 2 plaque assay was used to examine the potential synergistic and antagonistic effects of each additive on the apparent rate of HRP-mediated DAB oxidation. The same concentration of reagents as the ultraView ™ Detection Kit (VMSI 760-500: 253-4290, 253-4292 and 253-4293) were used in 1X Reaction Buffer ™ containing 0.1% fish gelatin for each assay. In each trial, the intensifiers were added together, one at a time. The results are summarized in Figure 9 and Table 2. TABLE 2: THE INFLUENCE OF INTENSIFIERS WAS ASSESSED IN THE APPARENT VMÁX FOR HRB OXIDED DAMAGE WHEN ADDED SEQUENTIALLY TO THE ULTRAVIEW ™ DETECTION KlT.

[240] The optical density of oxidized DAB was monitored at 455 nm. (NMO = 4-methyl-morpholine N-oxide).
[241] As previously shown in Table 1, 10 mM boric acid increased the apparent HRP rate by 265%. The addition of calcium chloride to HRP assays has been shown to increase the stability and apparent rate of HRP. The addition of both 10 mM calcium chloride and 10 mM boric acid to the assay containing 10 mM imidazole synergistically increased the apparent rate of HRP.
[242] Morpholine-A / -oxide increased the apparent rate of HRP reactions (see Table 1); however, when added to test mixture 4 (see Table 2, Entry 5), an antagonistic effect was observed. The addition of either 50 mM L-histidine or 10 mM pyrimidine to reaction mixture 5 (Table 2, Entries 6 and 7) increased the apparent rate of HRP. These data support the screening of the use of ZV-oxides in an intensification solution. Pyrimidine-N-oxide can be used in combination with L-histidine to increase DAB deposition in HCI tissue staining (see Figure 11). EXAMPLE 4 IHC TISSUE COLORING WITH INTENSIFIERS
[243] IHC staining was performed for bcl2 on tonsil tissue using enhanced DAB chromogen solutions to further examine synergistic enhancer effects on DAB deposition. A summary of pathology classification for IHC staining is shown in Table 3. Reader 1 performed all pathology assessments during the same period of time. Reader 2 performed the batch evaluations when the slides were initially produced and is responsible for some variability in the classification. TABLE 3: SUMMARY OF PATHOLOGY CLASSIFICATION FOR IHC BCI2 COLORING ON TONSILLA FABRIC USING DAB ULTRAVIEW ™ COLORING AND INTENSIFIED DAB CHROMOGEN SOLUTIONS.


[244] All compositions of the new base buffers contain 5.5 mM 3,3-diaminobenzidine tetrachloride (DAB-4 HCI) and 0.05% by weight of Brij® 35 (peroxidase free). [Base 1: 50 mM L-histidine (pH = 6.5); Base 2: 10 mM imidazole (pH = 6.5); Base 3: 2.43 mM citric acid, 5.13 mM sodium phosphate (pH = 5.3); Base 4: 10 mM L-histidine (pH = 6.5); Base 4a: 10 mM L-histidine (pH = 6.5), 10 mM calcium chloride, 10 mM boric acid.]
[245] Two general observations were noted. First, an apparently maximum rate intensification for DAB HRP deposition was achieved by combining 2-3 intensifying components. Additional intensifiers did not increase the signal intensity of the strongest DAB staining in the tissue; however, the percentage of cells stained with the highest signal strength increased through the tissue. This observation was largely due to the limited number of twists and turns observed by HRP-DAB oxidation reactions in the tissue. Second, the enhancers that increased HRP-mediated DAB oxidation in the plaque assay provided a more discrete deposition of DAB in the tissue. DAB staining was generally less diffuse.
[246] IHC staining of bcl2 (tonsil) using a formulated 5.5 mM DAB solution or with 10 mM imidazole or 10 mM L-histidine and 0.05% Brij® 35 is shown in Figures 10 and 11. The pathological review of DAB staining with 10 mM L-histidine showed an intensity similar to that produced with 10 mM imidazole.
[247] Staining tissue with 5.5 mM formulated DAB solutions or with 10 mM pyrimidine - / / - oxide or 10 mM 2-hydroxypyrimidine in 10 mM L-histidine and 0.05% Brij ® 35 is shown in Figures 12 and 13. The pathological review of DAB staining with both intensifier solutions showed that pyrimidine- / V-oxide provided the best DAB signal for background noise ratio for the two intensifiers.
[248] IHC staining of bcl2 in tonsil tissue was assessed using the addition of an "enhancement solution" to a standard ultraView ™ detection kit. No correction was made to the concentration of the ultraView ™ reagents to compensate for the dilution of the intensification solution (see Figures 14 to 17). 50 mM L-histidine and 10 mM pyrimidine (Figure 16) were collected by pathology reader 1 as a preferred DAB stain, as shown in Table 3. 100 mM imidazole and 50 mM boric acid increased DAB deposition , but reduced the dynamic range of the DAB signal and increased the serum background. A lower concentration of intensifiers would increase the dynamic range of the DAB signal. A 10 mM solution of L-histidine, 10 mM 2-hydroxypyridine, 10 mM calcium chloride, 10 mM boric acid showed a lower DAB signal when reformulating a DAB solution (bottom row of Table 3). 10 mM pyrimidine-A / -oxide is a prime candidate for an intensification solution. EXAMPLE 5 MICHAELIS-MENTEN KINETICS
[249] To further study the synergistic effect on the increased apparent Vmax for HRP oxidized DAB, the Michaelis-Menten kinetics was calculated for the best intensified DAB chromogen mixtures in Table 3. A 1:32 dilution of the multimer of HRP ultraView ™ was governed with a variable concentration of hydrogen peroxide (0.015 pM to 0.514 pM) to saturate the apparent HRP speed. Initially, both imidazole and L-histidine were examined with 10 mM pyrimidine (see Figure 18) and 10 mM 2-hydroxypyrimidine (see Figure 19). Similar Km values calculated at 1 / 4Vmax, but imidazole yielded an apparent Vmax higher than L-histidine (see Table 4). The definition of Vmax = kcat • [E] totai when the concentration of enzyme substrate was at saturation levels. When the enzyme concentration was kept constant, the apparent Vmax is proportional to kcat (the apparent turnaround for HRP or the first constant order rate). Imidazole increased the apparent HRP turnaround higher than L-histidine. TABLE 4: INFLUENCE OF INTENSIFIERS ON THE APPEARING VARIABLE FOR DAB OXIDED BY HRP WHEN COMBINED WITH 50 MM IMIDAZOLE, 10 MM CALCIUM CHLORIDE AND 10 MM BORIC ACID.

Km was determined to Wmax.
[250] Chromogen DAB imidazole solutions with intensifiers were screened to influence the apparent HRP Vmax (see Figure 20 and Table 4). 50 mM imidazole was used for further intensification of DAB oxidation. The intensification effect on the apparent HRP turnaround was 10 mM 2-hydroxypyridine> 10 mM pyrimidine-N-oxide> 50 mM pyrimidine> 10 mM 2-hydroxypyrimidine. These results were compared to those observed color intensities discussed in Table 3.
[251] Using DAB Chromogen solutions in Table 4, a plaque assay was performed using varying concentrations of the HRP ultraView ™ multimer (0.27 pg to 68.8 pg). Apparent Vmax was monitored and data reported as a percentage increase or decrease in apparent Vmax, compared to non-intensified reactions (see Table 5). An increase in HRP apparent Vmax was observed as the HRP concentration was decreased. The magnitude of change increased at lower HRP concentrations. These data confirmed the results of Table 4 in which imidazole provided a higher apparent Vmax compared to L-histidine. this effect was observed for a majority of concentrations. TABLE 5: INFLUENCE OF BUFFER SALES AND INTENSIFIERS ON THE APPARENT VMAX FOR HRB OXIDIZED DAB WHEN ADDED TO THE ULTRAVIEW DETECTION KlT ™.

[252] The optical density of oxidized DAB was monitored at 455 nm. [Intensifiers: (A) = 10 mM pyrimidine; (B) = 10 mM 2-hydroxypyrimidine, 10 mM calcium chloride, 10 mM boric acid; (C) = 10 mM 2-hydroxypyridine, 10 mM calcium chloride, 10 mM boric acid; (D) = 10 mM pyrimidine-N-oxide, 10 mM calcium chloride, 10 mM boric acid]. EXAMPLE 6 DEISH FABRIC COLORING WITH INTENSIFIERS
[253] ISH tissue staining was examined using enhanced DAB solutions. HER-2 3-in-1 mouse xenographs of HER-2 CaLu3, ZR-75-1 and MCF7 positive carcinoma cell lines were treated with a HER2 DNA probe (VMSI 480-4495) and stained with the chromogen solution DAB ultraView ™ or an enhanced DAB chromogen solution containing 10 mM L-histidine (see Figures 21 to 22). 10 mM L-histidine increased the deposition of DAB in the ISH staining of CaLu3 carcinoma cells that have an overexpression of HER2. The increased DAB deposition was marginal for the strongest ISH signals, but the signal strength for the weaker DAB signals was increased across the tissue. The same observation was made with deposition of DAB intensified in IHC. EXAMPLE 7 TSA FABRIC COLORING WITH INTENSIFIERS
[254] Enhanced DAB deposition was evaluated in a TSA-IHC stain of bcl2 tissue in tonsil tissue. The bcl2 antigen was stained with TSA using TA-HQ deposition for 4 minutes after a 16 minute incubation of the primary bcl2 Ab. The deposition of tiramide was performed with and without 10 mM of 2-hydroxypyrimidine. DAB deposition was performed with either the DAB ultraView ™ Chromogen solution or a DAB Chromogen solution that contains 10 mM L-histidine (see Figures 23 to 26). 10 mM L-histidine increased the deposition of DAB in the tissue.
[255] In a parallel study, the bcl2 antigen was stained with TSA using TA-HQ deposition for 4 minutes after an 8-minute incubation of the primary Ab of bcl2 (see Figures 27 and 28). 10 mM of 2-hydroxypyrimidine increased the deposition of tiramide and therefore increased the deposition of DAB. The percentage of staining of cells with DAB increased in areas with low expression of low bcl2 antigen. EXAMPLE 8 RIBOSSONDA COLORING 18s
[256] CaLu-3 xenograft tissues integrated with formalin-fixed paraffin were mounted on Superfrost® slides, deparaffinized and removed by antigen using RiboClear ™ denaturant (a component of the RiboMap® kit; Ventana® p / n 760-102) for 12 minutes of incubation, CC2 reagent (Ventana® p / n 950-123) and protease 3 for 8 minutes of incubation (Ventana® p / n 760-2020). Following removal, two drops (200 pl) of an 18s probe of NP haptenoid sense or antisense filament were dispensed on a slide, denatured at 80 ° C for 8 minutes and hybridized at 65 ° C for 6 hours. Following hybridization, the slides were washed 3 times using 0.1x SSC at 75 ° C for 8 minutes; where each haptenoylated NP probe was detected using 5 pg of a mouse anti-NP HRP conjugate per 100 pl of each 55 pl of HQ thyramide and H2O2 conjugate (component of the DAB Ventana® 760 ultraView ™ kit -500) and incubated for 12 minutes. The deposited tiramide-HQ was detected using 0.5 pg of a mouse anti-HQ HRP conjugate followed by a drop of DAB (5.5 mM DAB; 0.05% Brij® 35; 10 mM L- histidine; 10 mM 2-hydroxypyridine) and H2O2 incubating on the slide for 8 minutes. DAB ultraView ™ was used as the reference, after rinsing the slides in reaction buffer, 100 pl of copper (DAB ultraView ™ kit component) was applied to the slide for 4 minutes. The slides were countered using Hematoxylin II (Ventana® p / n 790-2208) and Bluing Reagent (Ventana® p / n 760-2037). The slides were dehydrated using gradient alcohols, covered with a slide and visualized using a bright field microscope. The comparison of the intensifier-treated sample and the ultraView ™ sample is shown in Figures 29 and 30, respectively. EXAMPLE 9 HPV COLORING
[257] C33, HeLa and CaSki xenografted fabrics integrated with formalin-fixed paraffin, were mounted on Superfrost slides, deparaffinized and removed by antigen using CC2 reagent (Ventana® p / n 950-123) and protease 3 for an incubation 8 minutes (Ventana® p / n 760-2020). Following removal, three drops (300 pl) of SISH Hyb Buffer (a component of the SISH ultraView ™ detection kit p / n 780-001) and three drops of DIG haptenylated HPV probe were dispensed on a slide, denatured at 75 ° C for 8 minutes and hybridized to 44 ° C for 6 hours. Following hybridization, the slides were washed 3 times using 0.1x SSC at 64 ° C for 8 minutes. The haptenoylated DIG probe was detected using 3 pg of a mouse anti-DIG HRP conjugate followed by DAB (5.5 mM DAB; 0.05% Brij® 35; 10 mM L-histidine; 10 mM 2-hydroxypyridine) and H2O2 (Ventana kit component of DAB ultraView ™ p / n 760-500) incubating on the slide for 8 minutes. DAB ultraView ™ was used as the reference. After rinsing the slides in the reaction buffer, 100 pl of copper (component of the DAB ultraView ™ kit) was applied to the slide for 4 minutes. The slides were stained using Hematoxylin II (Ventana® p / n 790-2208) and the Bluing reagent (Ventana® p / n760-2037). The slides were dehydrated using gradient alcohols, covered with a slide and visualized using a bright field microscope. The comparison of the intensifier-treated sample and the ultraView ™ sample is shown in Figures 31 versus 32, 33 versus 34 and 35 versus 36, respectively. EXAMPLE 10 DAB COLORING COMCD20
[258] Tonsil tissue integrated with fixed paraffin was mounted on Superfrost slides, dewaxed and had the antigen removed using CC1 batch reagent (Ventana® p / n 950-124). Following removal, a drop (100 pl) of UV inhibitor (a component of the ultraView ™ DAB kit) was dispensed on a slide and incubated for 8 minutes. Following the incubation of the inhibitor, 1 drop of mouse anti-CD20 (clone L-26; Ventana® p / n 760-2531) was dispensed on the slide and incubated for 16 minutes. Following 2 rinses with reaction buffer, the CD20 antibody was detected using 1 drop of the ultraView ™ universal HRP conjugate (a component of the ultraView ™ DAB kit) and incubated on the slide for 8 minutes. One drop of DAB (5.5 mM DAB; 0.05% Brij® 35; 10 mM L-histidine; 10 mM 2-hydroxypyridine) and H2O2 were each added to the slides and incubated for 8 minutes . The DAB ultraView ™ DAB was used as a reference. After rinsing, the slides in the reaction buffer, 100 pl of copper (component of the DAB ultraView ™ kit) were applied to the slide for 4 minutes. The slides were countered using Hematoxylin II (Ventana® p / n 790-2208) and Bluing's reagent (Ventana® p / n760-2037). The slides were dehydrated using gradient alcohols, laminated and visualized using a light-bottomed microscope. The comparison of the intensifier treated sample and the ultraView ™ sample is shown in Figures 37 and 38, respectively. EXAMPLE 11 DEAEC COLORING COMCD20 AND KI-67
[259] Tonsil tissue integrated with formalin-fixed paraffin was mounted on Superfrost slides, dewaxed and removed by antigen using CC1 batch reagent (Ventana® p / n 950-124). Following removal, a drop (100 pl) of Inhibitor (a component of the AEC Ventana® kit p / n 760-020) was dispensed on a slide and incubated for 8 minutes. Following the inhibitor incubation, 1 drop of mouse anti-CD20 (clone L-26; Ventana® p / n 760-2531) or rabbit anti-Ki67 (clone 30-9; Ventana® p / n 790-4286) was dispensed on the slide and incubated for 16 minutes. Following 2 rinses with reaction buffer, the antibody was detected using 1 drop of the universal HRP ultraView ™ conjugate (a component of the DAB ultraView ™ kit) and incubated on the slide for 8 minutes. One drop of an Intensifying Solution containing 50 mM L-histidine, pH 6.5, and 10 mM 2-hydroxypyridine was added and matched with one drop of each AEC and H2O2 and incubated for 8 minutes. A slide stained with AEC chromogen without any intensification was used as the reference. The slides were countered using Hematoxylin II (Ventana® p / n 790-2208) and Bluing's reagent (Ventana® p / n760-2037). The blades were allowed to air dry; were laminated with an aqueous mounting medium and visualized using a bright field microscope. The comparison of the intensifier-treated sample and the ultraView ™ sample is shown in Figures 39 versus 40, and 41 versus 42, respectively. EXAMPLE 12 DAB COLORING BY BCL-2
[260] Tonsil tissue integrated with formalin-fixed paraffin was mounted on Superfrost slides, dewaxed and had antigen removed using the CC1 batch reagent (Ventana® p / n 950-124). Following removal, a drop (100 pl) of UV inhibitor (a component of the DAB ultraView ™ kit) was dispensed on a slide and incubated for 8 minutes. Following the inhibitor incubation, 1 drop of mouse anti-bcl2 (clone 124; Ventana® p / n 790-4464) was dispensed on the slide and incubated for 16 minutes. Following 2 rinses with reaction buffer, the bcl2 antibody was detected using 1 drop of the universal HRP ultraView ™ conjugate (a component of the DAB ultraView ™ kit) and incubated on the slide for 8 minutes. One drop of DAB (5.5 mM DAB; 0.05% Brij® 35; plus any combination of enhancers investigated in Table 3) and H2O2 were each added to the slides and incubated for 8 minutes. DAB ultraView ™ was used as the reference, after rinsing the slides in the reaction buffer, 100 pl of copper (component of the DAB ultraView ™ kit) was applied to the slide for 4 minutes. The slides were countered using Hematoxylin II (Ventana® p / n 790-2208) and Bluing's reagent (Ventana p / n760-2037). The slides were dehydrated using gradient alcohols, laminated and visualized using a bright field microscope. EXAMPLE 13 DAB COLORING WITH BCL-2-TSA-HQ
[261] Tonsil fabric integrated with formalin-fixed paraffin was mounted on Superfrost slides, dewaxed and had antigen removed using the CC1 batch reagent (Ventana® p / n 950-124). Following removal, a drop (100 pl) of UV inhibitor (a component of the DAB ultraView ™ kit) was dispensed on a slide and incubated for 8 minutes. Following the incubation of the Inhibitor, 1 drop of mouse anti-bcl2 (clone 124; Ventana® p / n 790-4464) was dispensed on the slide and incubated for 16 minutes. Following 2 rinses with reaction buffer, the bcl2 antibody was detected using 1 drop of the ultraView ™ universal HRP conjugate (a component of the DAB ultraView ™ kit) and incubated on the slide for 8 minutes. 100 pl of each 55 µM tiramide-HQ with and without 10 mM of 2-hydroxypyridine and H2O2 (component of the DAB ultraView ™ Ventana® kit p / n 760-500) and incubated for 12 minutes. The deposited HQ-tiramide was detected using 0.5 pg of the mouse anti-HQ HQP conjugated followed by a drop of DAB (5.5 mM DAB; 0.05% Brij® 35; 10 mM L-histidine) and H2O2 incubated on the slide for 8 minutes. DAB ultraView ™ was used as the reference, after rinsing the slides in the reaction buffer, 100 pl of copper (component of the DAB ultraView ™ kit) was applied to the slide for 4 minutes. The slides were countered using Hematoxylin II (Ventana® p / n 790-2208) and Bluing's reagent (Ventana® p / n760-2037). The slides were dehydrated using gradient alcohols, laminated and visualized using a bright field microscope (Figures 23 to 26). EXAMPLE 14 DAB COLORING BY BCL-2-TSA-NP
[262] The tonsil tissue integrated with formalin-fixed paraffin was mounted on Superfrost slides, dewaxed and had antigen removed using the CC1 batch reagent (Ventana p / n 950-124). Following removal, a drop (100 pl) of UV inhibitor (a component of the DAB ultraView ™ kit) was dispensed on a slide and incubated for 8 minutes. Following the inhibitor incubation, 1 drop of mouse anti-bcl2 (clone 124; Ventana® p / n 790-4464), dilution of 1: 300, was dispensed on the slide and incubated for 16 minutes. Following the 2 rinses with reaction buffer, the bcl2 antibody was detected using 1 drop of the universal HRP ultraView ™ conjugate (a component of the DAB ultraView ™ kit) and incubated on the slide for 8 minutes. 100 pl of each 5 µM of tiramide-NP with and without 10 mM of 2-hydroxypyridine and H2O2 (component of the DAB ultraView ™ Ventana® kit p / n 760-500) and incubated for 12 minutes. The deposited HQ-tiramide was detected using 0.5 pg of the mouse anti-NP conjugated HRP followed by a drop of DAB (5.5 mM DAB; 0.05% Brij® 35; 10 mM L-histidine; 10 mM 2-hydroxypyridine) and H2O2 that incubates on the slide for 8 minutes. DAB ultraView ™ was used as the reference. After rinsing, the slides in the reaction buffer, 100 pl of copper (component of the DAB ultraView ™ kit) were applied to the slide for 4 minutes. The slides were stained using Hematoxylin II (Ventana p / n 790-2208) and Bluing's reagent (Ventana p / n760-2037). The slides were dehydrated using gradient alcohols, laminated and visualized using a bright field microscope (Figures 43 to 46).
[263] In view of the many possible embodiments to which the principles of the described invention can be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be considered as limiting the scope of the invention. Instead, the

权利要求:
Claims (9)
[0001]
1. METHOD FOR DETECTING A TARGET IN A SAMPLE, by means of proximal deposition of a marker, characterized by comprising: placing the sample in contact with a recognition solution, the recognition solution including a specific binding portion, which is specific to the target, wherein the specific binding portion comprises an antibody or a nucleic acid; labeling the specific binding portion with an enzyme, where the enzyme is an oxidoreductase or peroxidase; placing the sample in contact with a detection solution, the detection solution comprising an enzymatic substrate so that the marker is deposited in the vicinity of the target in the presence of a deposition intensifier that has the formula:
[0002]
2. METHOD according to claim 1, characterized by placing the sample in contact with a detection solution including enzymatically oxidizing the enzyme substrate using an oxidizing agent to form the marker, in which enzymatically oxidizing the enzyme substrate using a oxidizing agent includes reducing the solubility or stability of the enzyme substrate, so that the enzyme substrate becomes deposited as the marker.
[0003]
METHOD according to any one of claims 1 to 2, characterized in that the enzyme substrate is selected from 1,3-diaminobenzidine, 3-amino-9-ethylcarbazole, tetramethylbenzidine, a fluorescein, a luminophore, a coumarin, a BODIPY dye, a resorufin, a rhodamine, or a derivative thereof, or where the enzyme substrate is a tyramine derivative.
[0004]
METHOD, according to any one of claims 1 to 3, characterized in that the detection solution further comprises an accelerator selected from a heteroaryl compound, a boronic acid, a phenolic compound, or a combination thereof, optionally in which the heteroaryl compound is selected from imidazole, L-histidine, A / -pyridine oxide, A / -pyrimidine oxide, / V-methyl morpholine oxide and 2,2,6,6-tetramethylpiperidine-1-oxyl .
[0005]
5. METHOD according to any one of claims 1 to 4, characterized in that the detection solution further comprises a nonionic surfactant selected from a polyoxyethylene lauryl ether that has a formula (C2H4O) 23Ci2H2sOH; polyoxyethylene (20) sorbitan monoalkylate, the monoalkylate comprising between 8 and 14 carbons; a linear secondary alcohol polyoxyethylene having a formula C1-2H25-2gO [CH2CH2O] x, where x is equal to an integer between 2 and 12; and polyoxyethylene octyl phenyl ether or, wherein the detection solution further comprises an antioxidant selected from sodium bisulfate, sodium stannate, sodium metabisulfate and combinations thereof, or where the detection solution further comprises a salt containing Group I or Group II metal that has an MX2 or MX formula where M is a Group I or Group II metal selected from lithium, sodium, potassium, cesium, calcium, magnesium, strontium and barium; and X is selected from fluoride, chloride, bromide, iodide, carbonate, hydroxide and phosphate.
[0006]
6. KIT, characterized by comprising an enzyme, in which the enzyme is an oxidoreductase or peroxidase; a detection solution comprising a deposition intensifier and an enzyme substrate that produces a detectable portion after reacting with the enzyme, the deposition intensifier has the formula,
[0007]
KIT, according to claim 6, characterized in that the specific binding portion and the enzyme are linked together.
[0008]
KIT according to any one of claims 6 to 7, characterized in that the enzyme is peroxidase, in which it is horseradish peroxidase or glutathione peroxidase.
[0009]
9. METHOD according to any one of claims 1 to 5, or a kit according to any one of claims 6 to 8, characterized in that the deposition intensifier has a concentration ranging from about 5 mM to about 15 mM and the enzyme substrate has a concentration that ranges from greater than 0 mM to about 8 mM.

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-04| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-12-17| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-04-22| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-13| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/12/2011, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-12-15| B16C| Correction of notification of the grant|Free format text: REF. RPI 2597 DE 13/10/2020 QUANTO AO RELATORIO DESCRITIVO. |

优先权:

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申请号 | 申请日 | 专利标题

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US201061460349P| true| 2010-12-30|2010-12-30|

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US61/460,349|2010-12-30|

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PCT/US2011/067481|WO2012092322A1|2010-12-30|2011-12-28|Enhanced deposition of chromogens utilizing pyrimidine analogs|

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