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    • 专利摘要:
    methods of detecting monoclonal antibodies to enzymes that confer resistance to 2,4-dichlorophenoxyacetic acid in plants. the present invention relates to monoclonal antibodies and methods useful for determining and quantifying the presence of an aryloxy karyate dioxate enzyme.
    公开号:BR112012030788B1
    申请号:R112012030788-6
    申请日:2011-06-02
    公开日:2020-05-12
    发明作者:Guomin Shan；Gaofeng Lin；K.Smith-Drake Joelene；Marcelo J. Sosa
    申请人:Dow Agrosciences Llc；
    IPC主号:
    专利说明:

    Invention Patent Descriptive Report for MONOCLONAL ANTIBODY, HYBRIDOMA CELL LINE, AND METHODS FOR IDENTIFYING THE PRESENCE AND FOR QUANTITATIVE DETERMINATION OF AN AAD-12 ENZYME.
    Cross Reference to Related Order.
    [0001] This Application claims the benefit of Provisional Application No. US 61 / 351,593, filed on June 4, 2010, the disclosure of which is incorporated herein by reference in its entirety. Background of the Invention.
    [0002] 2,4-Dichlorophenoxyacetic acid (2,4-D) is in the herbicide phenoxyacid class and has been used in many monocot crops, such as corn, wheat and rice, for selective control of leafy weeds without damaging severely desired crop plants. 2,4-D is a synthetic derivative of auxin that acts to disrupt the hormonal homeostasis of normal cells and prevent balanced and controlled growth; however, the exact mode of action of this class of herbicides is not yet fully understood. Triclopir and fluroxypyr are pyridyloxyacetic acid herbicides that also act as a synthetic auxin.
    [0003] These herbicides have different levels of selectivity over certain plants (for example, dicotyledons are more sensitive than monocotyledons). Differentiated metabolism by different plants is an explanation for varying levels of selectivity. In general, plants metabolize 2,4-D slowly, so a variety of plant response to 2,4-D may be more likely explained by the different activities in the target sites (WSSA, 2002; Herbicide Handbook 8th edition, Weed Science Society of America; Lawrence, Kansas, p. 492). Plant metabolism of 2,4-D typically occurs via a two-phase mechanism, typically hydroxylation followed by conjugation with amino acids or glucose (WSSA, 2002).
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    2/25 [0004] Over time, certain microbial populations challenged with 2,4-D have developed an alternative pathway to degrade this xenobiotic that has resulted in complete 2,4-D mineralization. Successive herbicide applications select microbes that can use the herbicide as a source of carbon and energy for growth, giving them a competitive advantage in the soil. For this reason, the currently formulated 2,4-D has a relatively short half-life in the soil and no significant carry-over effect on subsequent harvests.
    [0005] One organism that was extensively investigated for its ability to degrade 2,4-D was Ralstonia eutropha (Streber, et al; 1987; Analysis, cloning, and high-level expression of 2,4dichlorophenixyacetic monooxygenase gene tfdA of Alcaligenes eutrophus JMP134. J. Bacteriol. 169: 2950-2955). The gene that encodes the enzyme in the initial stage of the mineralization path is tfdA. See U.S. Patent No. 6,153,401 and GENBANK Account No. M16730. The TfdA gene product catalyzes the conversion of 2,4-D to dichlorophenol (DCP) via a □ ketoglutarate-dependent dioxigenase reaction (Smejkal; et al .; 2001. Substrate specificity of chlorophenoxyalkanoic acid-degrading bacteria is not dependent upon phylogenetically related tfdA gene types. Biol. Fertil. Sols 33: 507-513). DCP has a low herbicidal activity compared to 2,4-D. TfdA has been used in transgenic plants to communicate resistance to 2,4-D in dicot plants, such as cotton and tobacco that are naturally sensitive to 2,4-D (Streber; et al .; 1989. Transgenic tobacco plants expressing a bacterial detoxifying enzyme are resistant to 2,4-D Bio / Technology 7:.. 811-816), and U.S. Patent No. 5,608,147).
    [0006] A large number of tfdA-like genes encoding enzymes capable of degrading 2,4-D have been isolated from soil bacteria
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    3/25 and their strings deposited in the Genbank database. Many tfdA homologues (amino acid identity> 85%) have enzyme properties similar to tfdA. However, there are several homologues that have a significantly lower identity to tfdA (25-50%), which still associate the characteristic residues with □ ketoglutarate dioxigenase Fe +2 dioxigenases. Thus, it is not obvious what are the substrate specificities of these divergent dioxygenases. [0007] A unique example with low tfdA homology (31% amino acid identity) is Delftia acidovorans sdpA (Kohler, HPE 1999; Delftia acidovorans MH: a versatile phenoxyalkanoic acid herbicide degrade ^ J. Ind Microbiol and Biotech. 23 : 336-340. Westendorf, et al .; 2002. The two enantiospecific dichlorprop / a-cetoglutaratodioxygenases from Delftia acidovorans MC1-protein and sequence data of Rdpa and SdpA. Microbiol. Res. 157: 317-22.). This enzyme has been shown to catalyze the first step in the mineralization of (S) dichloropropane acid (and other (S) -phenoxypropionic acid) as well as in the mineralization of 2,4-D (a phenoxyacetic acid) (Westendorfet et al .; 2003 Purification and characterization of the enantiospecific dioxygenases from Delftia acidovorans MC1 initiating the degradation of phenoxypropionates and phenoxyacetate herbicides (Acta Biotechnol. 23: 3-17).
    [0008] A plant gene for the codon-optimized aryloxyalkanoate dioxigenase, AAD-12, which encodes the enzyme originally isolated from Delftia acidovorans was first described for use as a herbicide resistance characteristic in WO 2007/053482, incorporated herein by reference . The characteristic confers tolerance to 2,4D and pyridyloxyacetate herbicides. The first report on processed soy that carries the AAD-12 gene was in United States Provisional Patent Application No. 61/263950, incorporated herein by reference.
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    4/25 [0009] Companies that develop and commercialize recombinant DNA traces to formulate products for planting seeds, implement and adhere to strict product management plans. These administration plans require the use of quantitative and qualitative protein detection methods validated for recombinant traits to accompany the introgression of traits and seed production activities, as well as the monitoring of grain harvest for that trait. These detection methods must be easy and robust enough to use under LPG and non-LPG conditions. In addition, the methods must be user friendly enough to be easily employed by farmers in the fields, corn dealers in the silos, and customs officials at the borders. Thus, robust, high-quality, easy-to-use protein detection methods and commercial kits are useful and necessary.
    [00010] Although immunoassays are well known in the art, the development of a robust, high-quality, validated ELISA (enzyme-linked immunosorbent assay) method that is reproducibly capable of detecting a given transgenic product in a sequence of tissue samples both in the laboratory and in rural settlements, they are neither trivial nor routine. Even more challenging is to find antibody pairs that are particularly adapted to the development of a lateral flow ELISA strip to detect a transgenic AAD12 event.
    Summary of the Invention.
    [00011] The present invention provides a panel of monoclonal antibodies (mAbs) and the hybridoma cell lines that produce them. The strains were deposited with the American Type Culture Collection under the terms of the Budapest Treaty. These
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    5/25 mAbs are surprisingly well adapted to detect a gene product from a transgenic AAD-12 event in various plants and plant tissues. The invention also provides quantitative and qualitative immunoassays using the immunoglobulins of the invention. Detailed Description.
    [00012] The present invention encompasses antibodies reactive with
    AAD-12 and the hybridomas that produce mAbs. The table below lists the hybridoma lineage designations and the corresponding designations deposited with the ATCC.
    Hybridoma / Designation of mAb Designation of the Depositat the ATCC Deposit Date atATCC 539B181.2 PTA-10919 May 5, 2010 539B470.2 PTA-10920 May 5, 2010 539B498.2 PTA-10921 May 5, 2010 539B304.2 PTA-10922 May 5, 2010 539B478.2 PTA-10923 May 5, 2010
    [00013] The present invention also includes methods of using mAbs to isolate or detect AAD-12 comprising: a) immobilizing said antibody on a surface; b) contacting said immobilized antibody with the mixture containing AAD-12; c) separating said immobilized antibody bound to AAD-12 from said mixture; and d) recovering the AAD12 by removing the AAD-12 bound to the antibody from said immobilized antibody.
    [00014] The present invention also includes a method for using the claimed antibodies to identify the presence of AAD-12 in a biological sample comprising: a) immobilizing said antibody on a test surface; b) contacting said test surface with a liquid suspected of containing the AAD-12, washing said test surface with a suitable solution; c) contact said test surface with an anti-AAD-12 antibody marked with a report group and wash said test surface with a convenient solution 870190091335, of 9/13/2019, p. 11/85
    6/25 te; d) detecting the presence of said report group.
    [00015] The invention additionally includes an analytical method for the quantitative determination of the AAD-12 enzyme expressed in transgenic plants, especially soybean and cotton plants. The AAD-12 protein is extracted from soy samples with a solution of PBS (phosphate buffered saline). The extract is centrifuged; the aqueous supernatant is collected and diluted. An aliquot of the diluted sample is incubated with the anti-AAD-12 monoclonal antibody conjugated to the enzyme in the wells of a microplate coated with the anti-AAD-12 polyclonal or monoclonal antibody in a sandwich ELISA. Both antibodies in the sandwich pair capture the AAD12 protein in the sample. At the end of the incubation period, unbound reagents are removed from the microplate by washing with PBS. The presence of AAD-12 is detected by incubating the enzyme conjugated with an enzyme substrate, generating a colored product. Since AAD-12 is bound in the antibody sandwich, the level of color development is proportional to the concentration of AAD-12 in the sample (that is, lower concentrations of protein result in weaker color development). The absorbance at 450 nm minus the absorbance at a reference wavelength (such as 650 nm) is measured using a microplate reader. A calibration curve is estimated from seven standard concentrations using a quadratic regression equation. This ELISA for AAD-12 is specific and quite sensitive for the quantification of AAD-12 in extracts from plant tissue samples. In addition, the antibodies of the invention can be used to confirm the presence of AAD-12 by using a standard western blotting procedure.
    [00016] The preparation of antibodies against the protein of interest is well known in the art. See Galfre and Milstein, Methods in Enzymology, volume 73, Academic Press, New York (1981); James W.
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    7/25
    Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, Florida (1986); Current Protocols in Molecular Biology, F. M. Ausubel, et al. editor, Wiley Interscience, New York, (1987).
    [00017] To prepare antibodies reactive with a protein of interest, the protein must first be enriched or purified. The relatively crude protein antigenic preparations can be used for immunization purposes. However, highly purified protein is needed to determine precisely whether hybridomas are producing the sought-after monoclonal antibodies or to analyze antibody titers of the immune serum.
    [00018] Once AAD-12 has been isolated, antibodies specific for AAD-12 can be developed by conventional methods that are well known in the art. Repeated injections into a chosen host over a period of weeks or months will elicit an immune response and result in significant titers of anti-AAD-12 serum. Preferred hosts are mammalian species and the most highly preferred species are rabbits, goats, sheep and rats. Blood drained from such immunized animals can be processed by established methods to obtain the antiserum (polyclonal antibodies) reactive with AAD-12. The antiserum can then be purified by affinity by adsorption to AAD-12 according to techniques known in the art. The affinity purified antiserum can also be purified by isolating the immunoglobulin fraction within the antiserum using procedures known in the art. The resulting material will be a heterogeneous population of immunoglobulins reactive to AAD-12.
    [00019] Anti-AAD-12 mAbs are rapidly prepared using purified AAD-12. The methods for producing mAbs have been practiced for several decades and are well known to those of ordinary skill in the art. Subcutaneous intraperitoneal injections
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    8/25 repetitions of AAD-12 in the adjuvant will elicit an immune response in most animals, especially in mice. Hyperimmunized B-lymphocytes are removed from the animal and fused with a cell line of suitable fusion partners capable of being cultured indefinitely. The numerous mammalian cell lines are convenient fusion partners for the production of hybridomas. Many of these strains are commercially available from ATCC and commercial suppliers.
    [00020] Once fused, the resulting hybridomas are grown in selective growth medium for one to two weeks. Two well-known selection systems are available to eliminate non-fused myeloma cells or fusions between myeloma cells from the mixed hybridoma culture. The choice of the selection system depends on the immunized rat litter and the myeloma fusion partner used. The AAT selection system, described by Taggart and Samloff, Science 219, 1228 (1982), can be used; however, the HAT selection system (hypoxanthine, aminopterin, thymidine), described by Littlefield, Science145, 709 (1964), is preferred because of its compatibility with rat cells and the aforementioned fusion partners.
    [00021] The growth medium used is then evaluated for the immunospecific mab secretion. The immunosorbent-linked enzyme testing procedures are best adapted for this purpose; although, radioimmunological tests adapted to the evaluation of large volumes are also acceptable. Multiple assessments designed to consecutively reduce the considerable number of inapplicable or less desired cultures should be performed to isolate the small percentage of mAbs in the present invention. Cultures that secrete AAD-12 reactive mAbs were isotyped using commercially available assays.
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    9/25 [00022] Hybridoma cultures that secrete the highly sought anti-AAD-12 mAbs should be subcloned several times to establish monoclonality and stability. Well-known methods for subcloning non-adherent eukaryotic cell cultures include techniques for restrictive dilution activated cell activation, mild agarose and fluorescence. After each subcloning, the resulting cultures should be re-analyzed for antibody and isotype secretion to ensure that a stable culture that secretes the antibody has been established.
    [00023] The claimed anti-AAD-12 antibodies can be immobilized on a surface so that a part of the antibody binding site remains exposed and is capable of binding with AAD-12. A wide variety of antibody immobilization schemes have been developed over the past few decades. Immobilization can be accomplished by covalently attaching the antibody directly to the desired surface or by bridging the antibody to the surface.
    [00024] CNBr and the carbodiimide binding of antibodies to polysaccharide trapped in beads, such as Sepharose ® (Pharmacia, Pistcataway, NJ) are illustrative of direct binding schemes that are compatible with the invention. Direct bindings generally do not target antibodies in any particular way; however, some types of direct bonds are able to reproducibly target the antibody to the immobilizing substance.
    [00025] Preferred binding schemes orient the antibody in such a way that its antigen binding regions remain exposed. One such scheme uses the natural carbohydrate found in the antibody's heavy chains. First by oxidizing the carbohydrate parts to the corresponding aldehydes, then reacting the aldehyde with a primary amino group on the surface,
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    10/25 bind the antibody in an advantageous orientation.
    [00026] Many types of bridges are possible and include small organic ligands that covalently bind the antibody to the immobilizing substance. Such spacer arms are acceptable and should preferably not interact with the protein once the bridge has been formed.
    [00027] The foregoing is in no way intended to limit the scope of the invention. Numerous other well-known schemes for binding antibodies to immobilizing substances are compatible with the invention.
    [00028] It is well known that antibodies labeled with a reporting group can be used to identify the presence of antigens in different media. Antibodies labeled with radioactive isotopes have been used for decades in radioimmunological assays to identify, with great precision and sensitivity, the presence of antigens in various biological fluids. More recently, enzyme labeled antibodies have been used as a substitute for more popular ELISA radiolabeled antibodies.
    [00029] The antibodies of the present invention can be attached to an immobilizing substance, such as a polystyrene well or particle and used in immunoassays to determine whether AAD-12 is present in a test sample. In this embodiment of the invention, a sample is contacted with the immunoaffinity surface and allowed to incubate. After a wash step, any AAD-12 that has bound to the immunoaffinity surface is detected by contacting the surface with another antibody of the invention labeled with a reporting group.
    [00030] The use of side flow strips or immunochromatographic strips with the claimed antibodies and the test methods are compatible with the invention. The lateral flow tests are well known
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    11/25 of those in the art. See for example US 6,485,982. In this mode, lateral flow tests can be used for the qualitative or semi-quantitative detection of AAD-12 alone or simultaneously with other analytes. Lateral flow tests are the simplest to use of all the test formats described here and are particularly useful in field settlements where plant material is quickly extracted into a solution and tested on a side flow strip. In this mode it is only necessary to place the side flow strip in a liquid sample or apply the liquid sample to the side flow strip and read the results after a predetermined time. All lateral flow tests must incorporate a process control line or a sample control line that is used to validate the test result. The appearance of two lines, in this way, indicates a positive result, while a valid negative test produces only the control line. If only the test line appears, or if no line appears, the test is invalid.
    [00031] A typical lateral flow test strip consists of four main components; a sample pad on which the test sample is applied, a conjugate pad containing antibodies of the present invention conjugated to colored particles (typically colloidal gold particles, or latex microspheres); a reaction membrane, such as a hydrophobic nitrocellulose or cellulose acetate membrane on which an antibody other than the invention is immobilized in a line across the membrane as a capture zone or test line; and, a tailings reservoir designed to drain the sample through the reaction membrane by capillary action. [00032] The components of the side flow strip are usually attached to an inert support material and can be presented in a simple strip format or in a plastic package with a sample door and reaction window showing the capture zones.
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    12/25 ra and control. In another mode of the test modality, a test sample suspected of containing AAD-12 is dried on a surface, forming an immobilized test sample. A labeled antibody of the invention is then contacted with the immobilized test sample and allowed to incubate. If the sample contains AAD-12, the labeled antibody will bind to the immobilized AAD-12. This method can also be done using an unlabeled antibody of the invention followed by a secondary labeled antibody that binds to an antibody of the invention that has already bound with AAD-12. After washing, the immobilized test sample is measured to detect the presence of any reporting group.
    [00033] Reporter groups are typically enzymes, such as alkaline phosphatase, wild radish peroxidase or beta-Dgalactosidase. Convenient substrates produce a change in color when reacted with the enzyme. Thus, color intensity measurements can be quantified using a spectrophotometer. If the reporting group is a radioactive isotope, a suitable gamma or beta detector can be used to quantify the reporting group. The intensity of the reporting group is directly correlated with the amount of AAD-12 in the test sample.
    [00034] The following examples will help to describe how the invention is practiced and will illustrate the characteristics of the antiAAD-12 antibodies and claimed assays.
    EXAMPLE 1.
    Immunogenic Preparation.
    [00035] AAD-12 protein was extracted from lyophilized leaf tissue taken from transgenic soy in a PBST (Saline phosphate buffer with 0.05% Tween 20, pH 7.4) to base buffer solution added with stabilizers , and soluble proteins were collected in the supernatant after centrifugation. The supernatant was easy
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    13/25 and the soluble protein was allowed to bind to the Fenil Sepharose ™ (PS) beads (GE Healthcare). After an hour of incubation, the PS beads were washed with PBST and the bound protein was eluted with Milli-Q ™ water. Sodium chloride was added to increase conductivity and the purified PS protein was loaded onto an anti-AAD-12 immunoaffinity column that had been conjugated to a specific polyclonal antibody AAD-12 raised against recombinant AAD-12 produced in Pseudomonas fluorescens . Unbound protein was collected from the column and the column was washed extensively with previously chilled PBS (saline phosphate buffer, pH 7.4). The bound protein was eluted from the column with a 3.5 M NaSCN buffer, 50 mM Tris ™, pH 8.0. AAD-12 derived from microorganisms and AAD-12 derived from soybeans were examined by SDS-PAGE and western blotting.
    [00036] In AAD-12 derived from microorganisms, the main protein band, as visualized on the SDS-PAGE gel stained with Coomassie, was approximately 32 kDa. As expected, the corresponding plant-derived AAD-12 protein derivative was identical in size to the microbial-derived protein. Predictably, the purified fractions of the plant contained less non-immunoreactive impurities than the AAD-12 protein. The copurified proteins were similarly retained in the column by weak interactions with the column matrix.
    [00037] The AAD-12 derived from microorganisms and the extract derived from plants showed a positive sign at the expected size in the Western blot using polyclonal anti-AAD-12 antibody. In the Western blot analysis of AAD-12, no immunoreactive proteins were observed in the negative control extract (native soybeans) and no proteins of alternating sizes (aggregates or degradation products) were observed in the samples of the transgenic plant.
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    14/25
    EXAMPLE 2.
    Hybridoma preparation.
    [00038] The mice were immunized with purified AAD-12, and standard fusion techniques were used to prepare a panel of hybridomas that express anti-AAD-12 monoclonal antibodies. Samples of the tissue culture media consumed were aseptically taken from each well containing a hybridoma culture and analyzed for reactivity to AAD-12 using the following antibody capture ELISA method. The microtiter wells were coated with a solution of 1 to 10 pg / ml of purified AAD-12. The wells were washed and samples of consumed tissue media were placed in the wells and allowed to incubate. The wells were washed and the anti-rat antiserum, anti-goat, labeled with wild radish peroxidase was added and allowed to incubate. The microplates were washed, the substrate was added to develop a color reaction and the microplates were read for OD (optical density). Wells with high DO readings were mapped back to the culture wells containing the hybridomas. Positive AAD-12 antibody cultures were continuously evaluated for antibody production to ensure growth stability and antibody production as the cultures expanded. Several cloning cycles by limiting dilution were performed to establish the true monoclonality of each culture. Additional assays on positive antibody clones were conducted to determine the suitability of each antibody for use in the quantitative detection methods presently claimed for use in the field with the plant material.
    EXAMPLE 3.
    Quantitative ELISA.
    [00039] This example is a method for quantitative determination
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    15/25 va of AAD-12 in soy tissues using antibodies and methods of the claimed invention. The quantitative range of the standard calibration curve is 0.25 ng / mL to 10 ng / mL in buffer. The level of AAD-12 soy protein in seeds, leaves (V5 and V10), root, and forage in stages R3 can be determined with a limit of quantification (LOQ) of 1.0 ng / mg and a limit of detection ( LOD) of 0.5 ng / mg.
    [00040] The test substances were representative soy tissue samples that were genetically modified to express the AAD-12 protein, and the non-transgenic control soybean of the Maverick variety. The tissues, listed below, were collected from the greenhouse.
    List of non-GM soy samples.
    Sample groupNo. Fabric Sample description 081008-001-0001 Forage (Whole plant; leaf and trunk; R3) non-transgenic control 081008-004-0001 Root (R3) non-transgenic control 081008-009-0001 Seed non-transgenic control 081008-010-0001 Sheets (V5) non-transgenic control 081008-011-0001 Sheets (V10) non-transgenic control
    List of transgenic soy samples.
    Sample groupNo. Fabric Sample description 081008-003-0001 Forage (Whole plant; leaf and trunk; R3) AAD-12 081008-006-0001 Root AAD-12 081008-007-0001 Sheets (R7) AAD-12 081008-012-0001 Seed AAD-12 081008-013-0001 Sheets (V5) AAD-12 081008-014-0001 Sheets (V10) AAD-12
    [00041] The reference substances used below in this study were a purified AAD-12 protein used as a standard
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    16/25 calibration and as a fortification material in the ELISA analysis, a purified AAD-12 protein, and a purified AAD-12 protein used in the cross-reactivity test.
    Protein Number of SubstancesTest Company Purity or Concentration Reference Cry1F 104301 0.164 mg / mL BIOT033236 AAD-1 105930 0.1805 mg / mL BIOT09-203007 Cry1Ac 102337 0.26 mg / mL BIOT08-162946 AAD-12 030732 0.2 mg / mL BIOT09-203009 PAT 105742 0.3 mg / mL BIOT063302 Cry35Ab1 104066 0.128 mg / mL BIOT08-162948 Cry34Ab1 104874 0.248 mg / ml BIOT09-203014
    [00042] All tests and reference substances were kept in monitored temperature freezers, and removed only for sample preparation and analysis. Briefly, the AAD-12 protein was extracted from soy samples (V5, V10, forage, and root) with PBST buffer (saline phosphate buffer solution containing 0.05% Tween ™ 20) with 0.75% ovalbumin ( OVA) (PBST / OVA). The AAD-12 protein was extracted from soybean seeds with a PBS solution containing 0.05% Tween ™ 20 (PBST) and 0.1% Triton ™ X-100. The extract was centrifuged, and the aqueous supernatant was collected, diluted and analyzed using specific ELISA for AAD-12. An aliquot of the diluted sample was incubated with the enzyme conjugated to the monoclonal anti-AAD-12 antibody 539B470.2 in the wells of a microplate coated with polyclonal anti-AAD-12 antibody in a sandwich ELISA. Both antibodies in the sandwich pair captured AAD-12 in the sample. At the end of the incubation period, unbound reagents were removed from the microplate by washing with PBST. The presence of AAD-12 was detected by incubating the antibody bound to the enzyme conjugate with an enzyme substrate, generating a colored product. How the AAD-12 was attached to the sandwich
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    17/25 of antibody, the level of color development was proportional to the concentration of AAD-12 in the sample (ie, lower concentrations of protein result in weaker color development). The color reaction was stopped by adding an acid solution and the absorbance at 450 nm minus the absorbance at 650 nm was measured using a microplate reader. A calibration curve was estimated from 7 standard concentrations using a quadratic regression equation with a coefficient of determination> 0.990. This ELISA for AAD-12 was highly specific for the quantification of the AAD-12 protein.
    EXAMPLE 4.
    Test Validation.
    [00043] The quantitative range of the method was preliminarily established independently during the development of the method and a pre-validation study. Standard concentrations gave the lowest average percentage errors of the given concentration points. The limit of detection (LOD) and the limit of quantification (LOQ) of the determination of AAD-12 in each tissue were empirically defined based on the test parameters (absorbance, background interference, and linear range of variation), interference matrices and / or doses constituting the standard curve. They were also supported by statistical approximations following the method of Keith et al. (Keith, LH, Crummett, W., Deegan, J., Junior, Libby, RA, Taylor, JK, Wentler, G. 1983. Principles of Environmental Analysis, Anal. Chem., 55, 22102218) and testing each sample of control fortified with 5 ng / mL (0.5 ng / mg) of AAD-12 protein.
    [00044] The cross-reactivity of that ELISA for AAD-12 for non-target proteins Cry1F, Cry1Ac, Cry34Ab1, Cry35Ab1, PAT and AAD1 was tested in this study. These proteins were prepared in concentrations of 1 pg / mL and 10 pg / mL in PBST / OVA. In the same mi
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    18/25 croplet, an AAD-12 standard curve was generated as a reference. The response OD of the non-target protein was interpolated from the AAD-12 standard curve and the percentage cross-reactivity was calculated using the following formula: cross-reactivity% = 100 x (AAD-12 measure conc. Of the standard curve / theoretical conc. target protein).
    [00045] The sample extracts (matrix) from each soybean tissue (1X, 5X and 10X dilutions) of the negative control were the peaks of different concentrations to create the standard curves. The standard curves of the peak matrix were interpolated from a standard curve without peaks running on the same microplate. A difference greater than 15% between the observed averages (a standard curve without peaks used to interpolate the standard concentrations of the peak matrix) and theoretical (concentration of the standard curve of the peak matrix) for each standard concentration level was considered indicative of a potential matrix effect.
    [00046] A series of five extractions was performed on transgenic soy tissues known to express AAD-12. Briefly, 1.5 ml of the buffer was added to the tissue sample (15 mg) and extracted as described above. Following extraction and centrifugation, the extracted solution was removed by pipette. After the first extract, a 200 pL aliquot of the buffer was added and mixed with the sample, centrifuged and the supernatant removed and added to the first extraction solution. Another 1.5 mL of the buffer was added to the tissue, and the extraction process was repeated. This procedure was repeated three more times to obtain 5 consecutive extractions. The concentration of AAD-12 in each extraction was determined using ELISA AAD-12. At least five replicates were studied for each tissue sample. The apparent efficiency of the tissue extraction process was determined by comparing the AAD12 protein in the first extract with the total AAD-12 protein in the five extracts.
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    19/25 [00047] The accuracy of the method was determined by measuring the recovery of AAD-12 protein from arrays of negative control peaks with low (0.5 ng / mg DW), medium (1, and 4 ng / mg DW) and high (8 ng / mg DW) levels of the AAD-12 protein. A minimum of five replicates of each concentration was analyzed. The accuracy of the assay was indicated as a percentage of recovery. Recoveries between 67 and 120% were considered acceptable.
    [00048] The accuracy of the method was determined using the results of fortified soybean control samples analyzed by two analysts over multiple days. The extracts from the control sample were fortified with three levels of the AAD-12 standard (0.25 ng / mg, 0.5 ng / mg, 4 ng / mg and 8 ng / mg). Each level of the fortified extract was run in triplicate on each ELISA microplate. The average recovery concentration, standard deviation (stdev), and percentage variation coefficient (% CV) were calculated for each of the samples.
    [00049] Positive samples (leaf V5 and forage (whole plant)) were also tested for accuracy. The predicted mean concentration, standard deviation (stdev), and percentage variation coefficient (% CV) were calculated for each sample. Precisions were calculated intra and inter days.
    [00050] The purpose of this experiment was to verify that the AAD-12 protein pattern and the AAD-12 protein in plant extracts exhibited a similar total response by ELISA. This was done for all transgenic tissues evaluating the agreement of the results of the dilution of a single extract interpolated from the quantitative range of the standard curve. The coefficient of variation of the interpolated results, of all quantifiable dilutions, was calculated for each type of tissue.
    [00051] The seeds, leaves, forage (whole plant) and root tissues were tested for false positive and false negative occurrences. Fifteen un-fortified control samples and fifteen fortified samples
    Petition 870190091335, of 9/13/2019, p. 25/85
    20/25 recorded at 0.25 ng / mg were analyzed for each tissue to determine false positive and false negative rates. A false and positive result occurs when the residue at, or above, the established LOD is found in a known analyte-free sample. A false negative occurs when no residue is detected in a sample fortified in the LOD.
    [00052] ELISA readings were recorded from a
    Molecular Dynamics microplate using the SOFTmax PRO program. The concentration data were transferred to SAS, JMP or Microsoft Excel for calculations of mean, percentage error, statistical mean, standard deviation, and% CV.
    [00053] The limit of detection (LOD) of an immunoassay is defined as the concentration of analyte that gives a response that has a statistically significant difference from the response of a zero analyte sample. The limit of quantification (LOQ), or working range of an assay, is generally defined as the highest and lowest concentrations that can be determined with an acceptable degree of accuracy. In this study, the LOD and LOQ aimed at determining AAD-12 in each tissue were empirically defined based on the test parameters (such as absorbance, background interference, signal to interference ratio, and linear range), interference matrices, and the standard curve concentrations. LOD and LOQ were also determined by standard statistical approximations. Following the established guidelines, LOD and LOQ were calculated using the standard deviation of the recovery results of 0.5 ng / mg. The LOQ was calculated as ten times the standard deviation (10s), and the LOD was calculated as three times the standard deviation (3s) of the analysis results for a minimum of 5 samples per matrix. The calculated results and the LODs and LOQs targets for each tissue are summarized in the table below.
    Petition 870190091335, of 9/13/2019, p. 26/85
    21/25
    Summary of LOD and LOQ Calculation of the ELISA for AAD-12 in Soy Tissue.
    Fabric Peak level ng / mg RecoveryMean ng / mg Standard deviation s 3 x s LODTarget ng / mg 10 x s LOQ Target ng / mg Fodder (Whole plant) 0.5 0.29 0.06 0.18 0.5 0.60 1.0 Root 0.5 0.30 0.05 0.15 0.5 0.50 1.0 Sheet V5 0.5 0.32 0.05 0.15 0.5 0.50 1.0 Seed 0.5 0.36 0.03 0.03 0.5 0.10 1.0 leafV10 0.5 0.46 0.03 0.03 0.5 0.10 1.0
    [00054] The target LOD is 0.5 ng / mg for all soybean matrices. O
    Target LOQ is 1.0 ng / mg for all soybean matrices.
    [00055] Several relevant proteins, such as Cry1F, Cry1Ac,
    Cry34Ab1, Cry35Ab1, PAT, and AAD-1 were tested for cross-reactivity. No cross-reactivity was observed at the concentrations tested for these proteins (10,000 ng / mL).
    [00056] The results of the matrix tests are summarized in the following table.
    Summary of Matrix Effects.
    Fabric P.15SGN # Matrix dilution a Lower dilution without matrix effect 1X 5X 10X Sheet V5 081008-010-0001 Yes Not Not 1: 5 Sheet V10-12 081008-011-0001 Yes Not Not 1: 5 Forage R3 (whole plant) 081008-001-0001 Yes Not Not 1: 5 Root R3 081008-004-0001 Not Not Not 1: 2 Seed R8 081008-009-0001 Yes Yes Not 1:10
    [00057] Yes represented that a standard curve is affected by the matrix when the average percentage error between the observed values and
    Petition 870190091335, of 9/13/2019, p. 27/85
    22/25 theorists of all seven levels of standard concentration is greater than 15%. It did not represent that any matrix effect or the average percentage error between the observed and theoretical values of all seven levels of standard concentration are less than 15%.
    [00058] A difference of greater than 15% between the observed and theoretical averages for any of the seven levels of standard concentration was considered indicative of a matrix effect. No matrix effects were observed at the root at level 1X of the peak matrix. No effect was found at the 5X level of the peak matrix for leaf V5, leaf V10 and forage (whole plant). However, the effects of the matrices were found in the seed at the 5X level. For AAD-12 quantification in soy tissues, at least a 2X dilution for the root is recommended; dilution of at least 5X is recommended for leaf V5, leaf V10 and forage; and a dilution of at least 10X for the seed is recommended.
    [00059] Determining the total level of AAD-12 protein in a sample is critical to examining the efficiency of extraction. Positive samples were extracted with buffer extraction for five consecutive times and the concentration of AAD-12 protein in each extract was determined by ELISA. The apparent efficiency of the extraction was based on the amount of AAD-12 protein in the first extraction in relation to the total sum of AAD-12 in the five extractions. The extraction efficiencies of AAD-12 protein from soy tissues are shown in the table below.
    Summary of EAD-12 Extraction Efficiency from Soy Tissue.
    Sample SGN # Average Extraction Efficiency (%) Standard deviation % CV % EE Range Forage (Whole Plant) 081008-003-0001 93.7 1.1 1.1 92.3-95.2 Root 081008-006-0001 90.0 0.4 0.5 89.6-90.6
    Petition 870190091335, of 9/13/2019, p. 28/85
    23/25
    Sheet V5 081008-013-0001 97.2 0.2 0.3 96.9-97.6 Seed 081008-012-0001 85.8 5.6 6.6 79.1-91.1 Sheet V10 081008-014-0001 93.3 1.4 1.5 91.1-94.7
    [00060] Extraction efficiencies for forage (whole plant), root, seed, leaf V5 and leaf V10 varied from 85.8 to 97.2%.
    [00061] The average levels of recovery of AAD-12 for all tissues when fortified to levels comparable to the LOQ, the midpoints and high points of the standard curve are shown in the table below.
    Summary of Precision Results.
    Matrix Fortification level ng / mg ng / mL a Recovery Rate (%) Average Range % CV n Lining- 8410.50.5 8040105 7170675 59-7760- 9.49.51 55552 gem -8 5-80 866 7957- 0.315.8 0 (Plant 7646- 14.4Entire) 7746-79 Root 8410.50.5 8040105 7271696 66-7764- 6.06.27, 55552-8 5-80 168 7662- 913,011 0 7651-7651-77 ,1leaf 8410.50.5 8040105 7576736 66-8067- 7,48,67, 55552 V5 -8 5-80 572 8366- 812,911 0 8253-7853-83 ,2Semen- 8410.50.5 8040105 7575747 72-7774- 2.41.62, 55552 you -8 5-80 374 7772-7671-7571-77 42.62.4 0 leaf 8410.50.5 8040105 9910096 97- 1.85.22, 55552 V10 -8 5-80 9397 10192-10594-9991-9491- 81.44.3 0 105a Samples were c 10X before analysis.
    [00062] The peaks at or above the LOQ, of the V5 sheet, of the
    V10 and seed were from 67 to 100% within the specification of 67
    Petition 870190091335, of 9/13/2019, p. 29/85
    24/25 to 120% for the average recovery with the percentage coefficients of variation (% CVs) at or below 16%.
    [00063] The accuracy and robustness of the test were examined using extracts of V5 leaf and forage (whole plant) containing four levels of the AAD-12 protein. The levels were 8 ng / mg, 4 ng / mg, 0.5 ng / mg and 0.25 ng / mg. The intraday precision of the assay was less than or equal to 6.3%, 10.8%, 9.6% and 15.0% of the V5 leaf extract fortified at 8, 4, 0.05 and 0.25 ng / mg , respectively. The intraday precision of the test was less than or equal to 3.5%, 13.1%, 10.1% and 10.9% of the forage extract (whole plant) fortified in 8, 4, 0.5 and 0.25 ng / mg, respectively. Leaf V5 and positive forage samples were also tested for the robustness of the test. The intraday precision of the assay was less than or equal to 9.7% and 19.7% for leaf V5 and the entire plant, respectively.
    [00064] The interassay accuracy across all days and analysts was 4.6%, 10.1%, 6.4% and 12.9% of the V5 leaf extracts fortified at 8, 4, 0.5 and 0 , 25 ng / mg, respectively. The accuracy of interassay across all days and analysts was 6.0%, 10.5%, 6.4% and 10.1% of forage extracts fortified at 8, 4, 0.5 and 0.25 ng / mg, respectively. The robustness of interassay across days and analysts was 11.3% and 14.1% for positive leaf V5 and forage, respectively.
    [00065] The equivalence of responses between the standard and the test substance in the ELISA for AAD-12 has been demonstrated using up to eight serial dilutions of AAD-12 positive tissue extracts. For each tissue extract, five or more of the dilutions fell within the quantitative range of the standard curve, and the% CV of the quantified results was less than 20%.
    [00066] Non-fortified control samples (blank spaces matrices) and samples fortified at 0.25 ng / mg (LOD = 0.5
    Petition 870190091335, of 9/13/2019, p. 30/85
    25/25 ng / mg) were analyzed to determine the false positive and false negative rates. There were no false positives in the un-fortified control samples and no reported false negatives from the fortified LOD samples analyzed in this study.
    [00067] In summary, the method has been validated over the concentration range of 1.0 to 8.0 ng / mg dry weight (DW) and has a validated limit of quantification (LOQ) in all soy tissues of 1 , 0 ng / mg DW and a limit of detection (LOD) in all soy tissues of 0.5 ng / mg DW. The AAD-12 protein was recovered at acceptable levels in all tissues. The validated assay is specific for the AAD-12 protein when compared to the non-target proteins tested in previous studies. For the quantification of AAD-12 protein in soy tissues, a dilution of 2X or greater is recommended for the root, dilution of 5X or greater is recommended for leaf V5, leaf V10 and forage, and a dilution of 10X or greater is recommended for the seed. In addition, the AAD-12 protein was efficiently extracted from all soy tissues. The assay was shown to have acceptable precision and accuracy, and no false positive or false negative results were seen below the LOD target. This AAD-12 ELISA method has been shown to be suitable for quantitative measurements of AAD-12 protein in soy tissues.
    权利要求:
    Claims (15)
    [1]
    1. Monoclonal antibody, characterized by the fact that it specifically binds to an aryloxyalkanoate dioxigenase enzyme (AAD-12), selected from the group of antibodies consisting of:
    539B181.2, produced by the hybridoma deposited with the ATCC as PTA-10919,
    539B470.2, produced by the hybridoma deposited with the ATCC as PTA-10920,
    539B498.2, produced by the hybridoma deposited with the ATCC as PTA-10921,
    539B304.2, produced by the hybridoma deposited with the ATCC as PTA-10922, and
    539B478.2, produced by the hybridoma deposited with the ATCC as PTA-10923.
    [2]
    2. Antibody, monoclonal according to claim 1, characterized by the fact that it is produced by the hybridoma deposited at the ATCC as PAT-10919, with a designation of 539B181.2.
    [3]
    3. Antibody, monoclonal according to claim 1, characterized by the fact that it is produced by the hybridoma deposited at the ATCC as PAT-10920, with a designation of 539B470.2.
    [4]
    4. Antibody, monoclonal according to claim 1, characterized by the fact that it is produced by the hybridoma deposited at the ATCC as PAT-10921, with a designation of 539B498.2.
    [5]
    5. Antibody, monoclonal according to claim 1, characterized by the fact that it is produced by the hybridoma deposited at the ATCC as PAT-10922, with a designation of 539B304.2.
    Petition 870190091335, of 9/13/2019, p. 32/85
    2/3
    [6]
    6. Antibody, monoclonal according to claim 1, characterized by the fact that it is produced by the hybridoma deposited at the ATCC as PAT-10923, with a designation of 539B478.2.
    [7]
    7. Hybridoma cell line, characterized by the fact that it produces a monoclonal antibody, as defined in any of claims 1 to 6, which is deposited with the American Type Culture Collection (ATCC) under accession numbers selected from the group consisting of in PTA-10919, PTA-10920, PTA-10921, PTA-10922 and PTA-10923.
    [8]
    8. Hybridoma cell line according to claim 7, characterized by the fact that it is deposited under the ATCC accession number PTA-10919.
    [9]
    9. Hybridoma cell line according to claim 7, characterized by the fact that it is deposited under the ATCC accession number PTA-10920.
    [10]
    10. Hybridoma cell line according to claim 7, characterized by the fact that it is deposited under the ATCC accession number PTA-10921.
    [11]
    11. Hybridoma cell line according to claim 7, characterized by the fact that it is deposited under the ATCC accession number PTA-10922.
    [12]
    12. Hybridoma cell line according to claim 7, characterized by the fact that it is deposited under the ATCC accession number PTA-10923.
    [13]
    13. Method for identifying the presence of an AAD-12 enzyme, characterized by the fact that it comprises:
    (a) immobilizing a first monoclonal antibody, as defined in any one of claims 1 to 6, on a test surface after washing said test surface;
    Petition 870190091335, of 9/13/2019, p. 33/85
    3/3 (b) contacting said test surface with a liquid suspected of containing AAD-12 for a period of time sufficient to permit bonding after washing said test surface;
    (c) contacting said test surface with a different second antibody, as defined in any one of claims 1 to 6, conjugated to a reporting group for a period of time sufficient to allow said second conjugated monoclonal antibody to bind after washing said test surface; and, (d) detecting the presence or absence of said report group.
    [14]
    14. Method for the quantitative determination of an AAD-12 enzyme, characterized by the fact that it comprises:
    (a) immobilize an AAD-12 antiserum on a test surface;
    (b) contacting said test surface with a liquid suspected of containing AAD-12 for a period of time sufficient to allow bonding after washing said test surface;
    (c) contacting said test surface with a different second antibody, as defined in any one of claims 1 to 6, conjugated to a reporting group for a period of time sufficient to allow said second conjugated monoclonal antibody to bind after washing said test surface; and, (d) quantifying the presence of said report group in comparison to a calibration curve.
    [15]
    15. Method according to claim 14, characterized by the fact that the conjugated monoclonal antibody is produced by the hybridoma deposited with the ATCC as PTA-10920.
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    US10609881B2|2018-08-03|2020-04-07|M.S. Technologies, L.L.C.|Soybean cultivar 74092327|
    US10542694B1|2018-08-03|2020-01-28|M.S. Technologies, L.L.C.|Soybean cultivar 77290232|
    US10537077B1|2018-08-03|2020-01-21|M.S. Technologies, L.L.C.|Soybean cultivar 76391606|
    US10492443B1|2018-08-03|2019-12-03|M.S. Technologies, L.L.C.|Soybean cultivar 70552824|
    US10631483B2|2018-08-03|2020-04-28|M.S. Technologies, L.L.C.|Soybean cultivar 63030535|
    US10492442B1|2018-08-03|2019-12-03|M.S. Technologies, L.L.C.|Soybean cultivar 64490328|
    US10631484B2|2018-08-03|2020-04-28|M.S. Technologies, L.L.C.|Soybean cultivar 60310209|
    US10477819B1|2018-08-06|2019-11-19|M.S. Technologies, L.L.C.|Soybean cultivar 75162339|
    US10631511B1|2019-04-04|2020-04-28|M.S. Technologies, L.L.C.|Soybean cultivar 83190332|
    US10667483B1|2019-04-22|2020-06-02|M.S. Technolgies, L.L.C.|Soybean cultivar 88092742|
    US10595486B1|2019-06-13|2020-03-24|M.S. Technologies, L.L.C.|Soybean cultivar 80330329|
    US10932431B1|2019-08-19|2021-03-02|M.S. Technologies, L.L.C.|Soybean cultivar 86072910|
    US10939651B1|2019-08-19|2021-03-09|M.S. Technologies, L.L.C.|Soybean cultivar 83011212|
    US10993403B2|2019-08-19|2021-05-04|M.S. Technologies, L.L.C.|Soybean cultivar 88042312|
    US10980204B2|2019-08-19|2021-04-20|M.S. Technologies, L.L.C.|Soybean cultivar 85010111|
    US10980206B2|2019-08-19|2021-04-20|M.S. Technologies, L.L.C.|Soybean cultivar 86052115|
    US10980205B2|2019-08-19|2021-04-20|M.S. Technologies, L.L.C.|Soybean cultivar 81140111|
    US10897866B1|2019-08-19|2021-01-26|M.S. Technologies, L.L.C.|Soybean cultivar 81442208|
    US10966396B2|2019-08-19|2021-04-06|M.S. Technologies, L.L.C.|Soybean cultivar 89192414|
    US10980207B2|2019-08-19|2021-04-20|M.S. Technologies, L.L.C.|Soybean cultivar 87242903|
    US10945400B1|2019-08-19|2021-03-16|M.S. Technologies, L.L.C.|Soybean cultivar 86220335|
    US10918064B1|2019-08-19|2021-02-16|M.S. Technologies, L.L.C.|Soybean cultivar 84322401|
    US10945401B1|2019-08-19|2021-03-16|M.S. Technologies, L.L.C.|Soybean cultivar 83372609|
    US10897867B1|2019-08-19|2021-01-26|M.S. Technologies, L.L.C.|Soybean cultivar 84450325|
    US11044870B2|2019-08-19|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 87011338|
    US10952394B2|2019-08-19|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 88390016|
    US10952395B2|2019-08-19|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 81371335|
    US10993402B2|2019-08-19|2021-05-04|M.S. Technologies, L.L.C.|Soybean cultivar 87390112|
    US10945399B1|2019-08-19|2021-03-16|M.S. Technologies, L.L.C.|Soybean cultivar 89442841|
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    US10945405B1|2019-08-20|2021-03-16|M.S. Technologies, L.L.C.|Soybean cultivar 81090603|
    US10945404B1|2019-08-20|2021-03-16|M.S. Technologies, L.L.C.|Soybean cultivar 83221630|
    US11044873B2|2019-08-20|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 88482541|
    US11219179B2|2019-08-20|2022-01-11|M.S. Technologies, L.L.C.|Soybean cultivar 87272833|
    US11212998B2|2019-08-20|2022-01-04|M.S. Technologies, L.L.C.|Soybean cultivar 88282833|
    US10952398B2|2019-08-20|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 84380724|
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    US10952399B2|2019-08-20|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 80230701|
    US10952397B2|2019-08-20|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 86160724|
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    US11172632B2|2019-08-20|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 85031644|
    US11044874B2|2019-08-20|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 83292541|
    US10932432B1|2019-08-20|2021-03-02|M.S. Technologies, L.L.C.|Soybean cultivar 87222215|
    US11044872B2|2019-08-20|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 84344663|
    US11044875B2|2019-08-20|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 81322943|
    US11044871B2|2019-08-20|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 84340383|
    US10952396B2|2019-08-20|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 88362310|
    US11006605B2|2019-08-27|2021-05-18|M.S. Technologies, L.L.C.|Soybean cultivar 84410120|
    US10993405B2|2019-08-27|2021-05-04|M.S. Technologies, L.L.C.|Soybean cultivar 85281832|
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    US11006604B2|2019-08-27|2021-05-18|M.S. Technologies, L.L.C.|Soybean cultivar 82431018|
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    US10952401B1|2019-08-28|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 83292238|
    US10999999B2|2019-08-28|2021-05-11|M.S. Technologies, L.L.C.|Soybean cultivar 83392343|
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    US11071274B2|2019-08-28|2021-07-27|M.S. Technologies, L.L.C.|Soybean cultivar 83050118|
    US10986797B2|2019-08-28|2021-04-27|M.S. Technologies, L.L.C.|Soybean cultivar 86440139|
    US11006606B2|2019-08-28|2021-05-18|M.S. Technologies, L.L.C.|Soybean cultivar 81201100|
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    US11006607B2|2019-08-29|2021-05-18|M.S. Technologies, L.L.C.|Soybean cultivar 80462534|
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    US10925245B1|2019-08-29|2021-02-23|M.S. Technologies, L.L.C.|Soybean cultivar 89021021|
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    US10905082B1|2019-08-29|2021-02-02|M.S. Technologies, L.L.C.|Soybean cultivar 82212235|
    US11140845B2|2019-08-29|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 88041740|
    US11134635B2|2019-08-29|2021-10-05|M.S. Technologies, L.L.C.|Soybean cultivar 82371519|
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    US10952403B1|2019-08-29|2021-03-23|M.S. Technologies, L.L.C.|Soybean cultivar 80462430|
    US11044879B2|2019-08-29|2021-06-29|M.S. Technologies, L.L.C.|Soybean cultivar 82352802|
    US11140847B2|2019-08-29|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 80372223|
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    US11013198B2|2019-08-29|2021-05-25|M.S. Technologies, L.L.C.|Soybean cultivar 84042612|
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    US11076556B2|2019-08-29|2021-08-03|M.S. Technologies, L.L.C.|Soybean cultivar 83422133|
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    US11076562B1|2020-02-13|2021-08-03|M.S. Technologies, L.L.C.|Soybean cultivar 95130401|
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    US11191238B2|2020-02-13|2021-12-07|M.S. Technologies, L.L.C.|Soybean cultivar 99240189|
    US11140855B2|2020-02-13|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 90420357|
    US11202427B2|2020-02-13|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 94110636|
    US11076563B1|2020-02-13|2021-08-03|M.S. Technologies, L.L.C.|Soybean cultivar 94040702|
    US11134640B2|2020-02-13|2021-10-05|M.S. Technologies, L.L.C.|Soybean cultivar 94220034|
    US11051481B1|2020-02-13|2021-07-06|M.S. Technologies, L.L.C.|Soybean cultivar 93020437|
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    US11051480B1|2020-02-13|2021-07-06|M.S. Technologies, L.L.C.|Soybean cultivar 93230440|
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    US11109558B1|2020-02-21|2021-09-07|M.S. Technologies, L.L.C.|Soybean cultivar 91220032|
    US11140856B2|2020-02-21|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 93440976|
    US11172639B1|2020-06-02|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 99310382|
    US11166430B1|2020-06-02|2021-11-09|M.S. Technologies, L.L.C.|Soybean cultivar 99120525|
    US11213001B2|2020-06-02|2022-01-04|M.S. Technologies, L.L.C.|Soybean cultivar 98320614|
    US11140857B1|2020-06-02|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 93320341|
    US11102951B1|2020-06-02|2021-08-31|M.S. Technologies, L.L.C.|Soybean cultivar 96130264|
    US11122765B1|2020-06-02|2021-09-21|M.S. Technologies, L.L.C.|Soybean cultivar 98220804|
    US11172637B1|2020-06-02|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 96350326|
    US11172640B1|2020-06-02|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 91230357|
    US11122766B1|2020-06-02|2021-09-21|M.S. Technologies, L.L.C.|Soybean cultivar 96140088|
    US11116169B1|2020-06-02|2021-09-14|M.S. Technologies, L.L.C.|Soybean cultivar 92050703|
    US11172638B1|2020-06-02|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 97320638|
    US11202429B1|2020-06-02|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 91410746|
    US11122764B1|2020-06-02|2021-09-21|M.S. Technologies, L.L.C.|Soybean cultivar 91210322|
    US11202430B1|2020-06-02|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 93120753|
    US11191240B1|2020-07-14|2021-12-07|M.S. Technologies, L.L.C.|Soybean cultivar 91210615|
    US11197452B1|2020-07-14|2021-12-14|M.S. Technologies, L.L.C.|Soybean cultivar 92140814|
    US11172642B1|2020-07-14|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 99150287|
    US11172641B1|2020-07-14|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 90140287|
    US11134642B1|2020-07-14|2021-10-05|M.S. Technologies, L.L.C.|Soybean cultivar 98272614|
    US11202433B1|2020-07-14|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 92010858|
    US11116170B1|2020-07-14|2021-09-14|M.S. Technologies, L.L.C.|Soybean cultivar 91250440|
    US11191241B1|2020-07-14|2021-12-07|M.S. Technologies, L.L.C.|Soybean cultivar 92220615|
    US11191242B1|2020-07-14|2021-12-07|M.S. Technologies, L.L.C.|Soybean cultivar 95450804|
    US11172643B1|2020-07-14|2021-11-16|M.S. Technologies, L.L.C.|Soybean cultivar 94440162|
    US11202432B1|2020-07-14|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 91410530|
    US11140858B1|2020-07-14|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 91040342|
    US11252913B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 97240377|
    US11202434B1|2020-07-17|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 93140657|
    US11252912B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 90220377|
    US11252914B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 95111047|
    US11202435B1|2020-07-17|2021-12-21|M.S. Technologies, L.L.C.|Soybean cultivar 92040765|
    US11219184B1|2020-07-17|2022-01-11|M.S. Technologies, L.L.C.|Soybean cultivar 95250357|
    US11252908B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 98310437|
    US11252909B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 94120737|
    US11252910B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 99350737|
    US11224183B1|2020-07-17|2022-01-18|M.S. Technologies, L.L.C.|Soybean cultivar 95040275|
    US11252911B2|2020-07-17|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 91110447|
    US11259490B2|2020-07-17|2022-03-01|M.S. Technologies, L.L.C.|Soybean cultivar 99250287|
    US11219185B1|2020-07-29|2022-01-11|M.S. Technologies, L.L.C.|Soybean cultivar 97250069|
    US11266103B2|2020-07-29|2022-03-08|M.S. Technologies, L.L.C.|Soybean cultivar 99150754|
    US11197453B1|2020-07-29|2021-12-14|M.S. Technologies, L.L.C.|Soybean cultivar 95130716|
    US11219186B1|2020-07-29|2022-01-11|M.S. Technologies, L.L.C.|Soybean cultivar 91120809|
    US11266102B2|2020-07-29|2022-03-08|M.S. Technologies, L.L.C.|Soybean cultivar 92220922|
    US11266101B2|2020-07-29|2022-03-08|M.S. Technologies, L.L.C.|Soybean cultivar 96310052|
    US11229179B1|2020-07-29|2022-01-25|M.S. Technologies, L.L.C.|Soybean cultivar 91410830|
    US11178837B1|2020-07-29|2021-11-23|M.S. Technologies, L.L.C.|Soybean cultivar 92230102|
    US11140860B1|2020-07-29|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 96220972|
    US11219187B1|2020-07-29|2022-01-11|M.S. Technologies, L.L.C.|Soybean cultivar 94240013|
    US11259491B2|2020-07-29|2022-03-01|M.S. Technologies, L.L.C.|Soybean cultivar 80540918|
    US11266104B2|2020-07-29|2022-03-08|M.S. Technologies, L.L.C.|Soybean cultivar 99030547|
    US11178838B1|2020-07-29|2021-11-23|M.S. Technologies, L.L.C.|Soybean cultivar 99262713|
    US11134643B1|2020-07-29|2021-10-05|M.S. Technologies, L.L.C.|Soybean cultivar 93410922|
    US11224184B1|2020-07-29|2022-01-18|M.S. Technologies, L.L.C.|Soybean cultivar 93330609|
    US11140859B1|2020-07-29|2021-10-12|M.S. Technologies, L.L.C.|Soybean cultivar 90120947|
    US11252915B1|2020-07-29|2022-02-22|M.S. Technologies, L.L.C.|Soybean cultivar 99040204|
    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-07-16| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|
    2019-12-10| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2020-03-10| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-05-12| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 02/06/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    US35159310P| true| 2010-06-04|2010-06-04|
    US61/351,593|2010-06-04|
    PCT/US2011/038848|WO2011153300A2|2010-06-04|2011-06-02|Monoclonal antibodies detection methods for enzymes that confer resistance to 2,4-dichlorophenoxyacetic acid in plants|
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