Test for diagnostics of analysts in a sample

专利摘要:

公开号:DK200500290U1
申请号:DK200500290U
申请日:2005-12-19
公开日:2006-04-28
发明作者:Charlton David
申请人:Advantage Diagnostics Corp；
IPC主号:

专利说明:

DK 2005 00290 U4
Test strip for diagnostic analysis of analyzers in a sample
BACKGROUND OF THE INVENTION Field of the Invention
The present invention relates to a rapid visual test with lateral flow for detecting analyte in a sample.
Description of Related Art
Lateral flow test devices are known in the art and are used in clinical diagnostics to determine the presence of an analyte of interest in a sample, such as a body fluid. However, such devices typically require a relatively large sample volume and large amounts of conjugate. They have also been associated with long waiting times before the results of the test can be read.
US 20010023076 discloses a test device containing a sample receiving zone located upstream of a reaction zone. The sample may migrate laterally toward the sample receiving end. US 5656503 discloses a conventional test strip in which two different antibodies are used to identify the same antigen. Thereby, the sample receiving end must be located upstream of a conjugate region, in order to obtain a unidirectional flow of sample fluid sequentially across a first antibody region and subsequently a second antibody region.
Brief Description of the Invention
According to one aspect, an immunodiagnostic test strip is provided for detecting an analyte of interest in a sample. According to some embodiments, the assay is an antibody such as an anti-human immunodeficiency virus (HIV) antibody or an anti-hepatitis C virus (HCV) antibody. In other embodiments, the analyte is an antigen such as a human chorionic gonadotropin (hCG).
Preferably, the diagnostic test strip comprises a conjugate source region comprising a conjugate designed to bind the analyte subject of interest; a sample application area disposed downstream of the conjugate source region and configured to receive the liquid sample; as well as a test area. The test region is located downstream of the conjugate source region and comprises an immobilized binder capable of binding specifically to the relevant analyte. For example, if the relevant assay is an antibody, the positive test region preferably comprises immobilized antigen. If the relevant assay is an antigen, the positive test region preferably comprises an immobilized antibody. The conjugate comprises a first binding component capable of binding the relevant analyte and a second visualization component.
The sample used in the test may be any liquid. Preferred samples include e.g. blood, serum, plasma, saliva and urine. The sample is applied to the positive test region and the conjugate is subsequently flowed from the conjugate source region. The conjugate flows across the membrane and comes into contact with any analyzer that may be bound to the positive test region, thereby producing a visual signal.
According to a preferred embodiment, the test strip comprises a test area with immobilized HIV antigen and is used to detect anti-HIV antibodies. In this embodiment, the test is useful for diagnosing whether a patient has HIV infection. According to another embodiment, the test strip comprises a test region with an immobilized HCV antigen and is used to detect anti-HCV antibodies in a sample. In this embodiment, the test is suitable for diagnosing whether a patient has HCV infection. According to a further embodiment, the test strip comprises a test region with immobilized anti-hCG antibodies. In that embodiment, the test is well suited to determine if an individual is pregnant.
The conjugate source region preferably comprises dried conjugate. For example, the conjugate source region may be a conjugate pad upon which the conjugate is dried. The conjugate pad is preferably in flow communication with the membrane. In some embodiments, the conjugate pad is moved to flow with the membrane after adding the sample to the sample application area. According to another embodiment, the conjugate source region is part of the membrane on which the conjugate is dried.
Dried conjugate is preferably resuspended and brought to flow by contacting the conjugate source region with wash buffer. According to one embodiment, the wash buffer comprises approx. 0.1% blocking agent, preferably a blocking protein such as casein. The wash buffer also preferably comprises approx. 0.1% detergent, preferably a nonionic detergent such as Triton X100 ™. According to one embodiment, the wash buffer comprises approx. 0.025% HEPES, approx. 0.85% sodium chloride, approx. 0.1% EDTA, approx. 1% mannitol, approx. 0.1% casein and approx. 1% Triton X100 ™ Detergent.
According to a further embodiment, the conjugate is dissolved in a buffer. The conjugate is caused to flow to the test region by adding the buffer comprising the conjugate to the conjugate source region.
According to a particular embodiment, the conjugate comprises protein A conjugated to colloidal gold. In other embodiments, the conjugate comprises an antibody, preferably a monoclonal antibody conjugated to colloidal gold. Preferably, the colloidal gold has a particle size of between approx. 20 and 80 nanometers.
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According to a further embodiment, the test strip further comprises a control area located on the membrane downstream of the test area. The control region preferably comprises a control binder capable of binding the conjugate. For example, if the relevant analyte is an antibody and the conjugate comprises an antibody binder such as protein A, the control binder may be rabbit IgG. If the relevant assay is an antigen and the conjugate comprises a monoclonal antibody specific for the antigen, the control binder may e.g. be protein A.
The test strip may comprise one or more additional components. A buffer pad may be in flow communication with the conjugate source region, so that adding wash buffer to the buffer pad causes the conjugate to flow from the conjugate source region and across the membrane to contact the test region. An absorbent strip and absorbent pad may be located downstream of the membrane to absorb excess buffer as it passes across the membrane. A desiccant tablet may also be disposed near the membrane to absorb excess liquid and moisture.
Optionally, the test strip may be provided in a housing such as a plastic housing. The housing preferably comprises a sample window located above the test and control areas as well as a buffer window located upstream of the test area.
The test strips of the present invention have several advantages over prior art. They enable detection of analyzers using relatively smaller sample volume as well as smaller amount of conjugate. They also enable the reading of test result after a shorter time.
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Brief description of the drawing
FIG. 1 shows a test card according to an aspect of the present invention.
FIG. 2 shows a top view of a test card according to one aspect of the invention.
FIG. 3 shows a side view of a test card according to one aspect of the invention.
Fig. 4 shows a sectional view of the components constituting a test strip according to one embodiment;
FIG. 5 shows a test result with a positive result indicating that the relevant analyte was detected in the sample.
FIG. 6 shows a test result with a negative result indicating that the relevant analyte was not detected in the sample.
FIG. 7 shows a test strip with a test cup according to a further aspect of the invention.
Detailed description of preferred embodiments
Diagnostic devices are provided to quickly determine if an analyte is present in a sample. Although the present invention is generally described as an immunodiagnostic device for detecting an antibody or antigen in a sample, those skilled in the art will readily understand that it can be readily modified to detect any relevant assay.
Thus far, immunodiagnostic devices have typically involved initial mixing of a conjugate with the sample to be assayed for the presence of an antibody. For example, in conjunction with some devices, dried conjugate was to be dissolved by the addition of a liquid sample, whereby the sample and conjugate could be premixed. The antibody to be detected, if present, will bind to the conjugate and visualized at a downstream test region comprising an immobilized antigen. However, in this system, a non-specific interaction occurred between the conjugate and other antibodies, or other compounds in the sample led to suppression of the conjugate signal. As a result, a large amount of conjugate was needed. Due to the large amount of conjugate used, the reaction was slow to become clear, thereby prolonging the period needed to obtain the test result. In addition, the sensitivity of the test was such that a significant amount of antigen was required at the test site to produce a visible positive signal.
The present invention overcomes the problems of the prior art in that the test area is brought into direct contact with the sample, whereby any relevant analyte can bind specifically to immobilized binder. For example, a relevant antibody may bind to immobilized antigen. Subsequently, conjugate is delivered to the test solution with buffer solution. The solvent front of the buffer solution does not eject bound compounds at the test region, thereby reducing non-specific interactions with the conjugate. As a result, the amount of conjugate required is significantly reduced and the rate of reaction is thereby increased. In addition, the sensitivity of the reaction is increased, which makes it possible to use less antigen at the test site.
The disclosed devices may be used, for example, to diagnose a disease or disorder in a patient such as human immunodeficiency virus (HIV) or hepatitis virus. They can also be used to determine whether an individual is pregnant. Other uses will be appreciated by those skilled in the art by reading the following.
definitions
Unless otherwise defined, all technical and scientific terms used herein will have the same meaning as will ordinarily be understood by those skilled in the art within the scope of the present invention. All methods, devices and materials which may be similar or equivalent to those described herein may be used in the practice of the present invention.
A "relevant assay" is any molecule or compound whose occurrence is to be identified in a sample, which include, without limitation: viral antigens, bacterial antigens, hormones such as insulin, follicle stimulating hormone (FSH), thyrotropin, relaxin, somatotropin and gonadotropin, enzymes, immunoglobulins, cytokines, drugs, cancer antigens, antigenic polysaccharides and nucleic acids Other analyzers which can be identified in samples using the methods provided and the apparatus provided will be apparent to those of ordinary skill in the art. Relevant assays include anti-HIV antibodies, anti-HCV antibodies, and human chorionic gonadotropin (hCG).
The term "conjugate" refers to a composition which is designed to bind to a relevant analyte and produce a detectable signal. The conjugate typically comprises a binding component conjugated to a label. The binding component allows the conjugate to bind to the relevant analyte and optionally to a The label produces a detectable signal, preferably a visual signal. In one embodiment, the visual signal is generated only after the conjugate binds to the analyte. In a preferred embodiment, the relevant analyte is an antibody and the conjugate comprises a binding component which is For example, the first binding component may be protein A. although other molecules capable of binding antibodies may be used. In another embodiment, the relevant assay is an antibody and the binding component of the conjugate comprises a antibody capable of binding anti Preferably, the antibody is a monoclonal antibody.
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The label is any molecule or compound which can be attached to or conjugated to the binding component and which can produce a detectable signal. A preferred label is colloidal gold. Alternatively, the label may be e.g. colored latex particles, colloidal silver or other colloidal metals, colloidal black, or other components known in the art. Preferably, the label has such a particle size that it does not interfere with the ability of the binding component to bind the relevant analyte. For example, when the binding component is protein A, the label has a preferred particle size of from about. 5 to approx. 120 nanometers, more preferably from ca. 20 to approx. 80 nanometers.
The conjugate is preferably prepared in a "conjugate buffer" which can stabilize and retain the conjugate. According to one embodiment, after preparation in conjugate buffer, the conjugate can be dried on a "conjugate pad", which will be described in more detail below. Conjugate dried on a substrate is considered to be "diffusion bound." The conjugate is "diffusion bonded". if it is capable of diffusing or flowing, for example by contacting a buffer, for example, conjugate which has been diffusion bound to the conjugate source region upon drying can be made soluble with buffer and thus to flow along the membrane of the test strip.
A "control conjugate" is a conjugate wherein the binding component is specific to a control component. A control conjugate is preferably used when it is probable that the entire conjugate will be bound to the assay on the test line and not available as a control. which is known to be present in the sample under analysis and capable of binding the control binder on the control strip, optionally the binding component of the control conjugate may be specific to a compound located in the control membrane's control region.
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The conjugate is preferably present in a "conjugate source region." The conjugate source region typically comprises conjugate which is diffusion bound thereto, such as dried conjugate. Alternatively, the conjugate source region may comprise conjugate dissolved in buffer solution such as In a further embodiment, the conjugate source region is a portion of the test membrane upon which the conjugate has been dried. In a further embodiment, the conjugate source region is a buffer pad to which buffer comprising conjugate may be added which is in fluid flow. The conjugate source region is typically located upstream of the test region on the test membrane, so that after the application of a solution comprising the conjugate or after the diffusion-bound conjugate has been made up, soluble, flowing across the test membrane to contact the test membrane.
The conjugate is preferably made soluble with "wash buffer" as described in more detail below. Briefly, wash buffer is a buffer solution preferably comprising a "blocking agent." Blocking agents are well known in the art and include any molecule or compound that reduces non-specific interactions such as non-specific antibody binding. are proteins such as casein and bovine serum albumin (BSA). Other useful blocking agents are commercially available and will be apparent to those of ordinary skill in the art.
"Sample buffer" is a buffer solution which preferably does not include conjugate. In some embodiments, the sample buffer comprises a blocking agent, while in other embodiments it does not. Sample buffer may optionally be applied to the membrane prior to addition to the sample, as will be described in more detail below.
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A "test membrane" is a solid support comprising a test area and optionally a control area. The sample to be analyzed is typically applied directly to the test membrane so that it contacts the test area and the control area. The test membrane may be any solid support to which an analyte binder and a control binder may be attached. Preferably, the test membrane is nitrocellulose.
The test membrane provides a lateral flow path for liquids. The test diaphragm and other components of the test strip are said to be in "flowing compound" if liquid, such as a liquid sample or buffer, can flow from one component to another. If the components of the test strip are in contact with each other, they are in flowing compound. It will be appreciated by those skilled in the art that direct contact between two specific components, however, is not a prerequisite for fluid connection.
"Test range", "test strip" and "test line" are used in the same meaning and refer to an area of the test membrane to which an analyte binder is attached.
An "analyte binding agent", "specific binding partner" or "binding component" is any molecule or compound designed to bind specifically to a relevant analyte. By way of example and not limitation, the analyte binding agent may comprise antigens, antibodies. , receptors, other polypeptides, peptides, haptenes, lectins, nucleic acid, including oligonucleotides, or small molecules. In one embodiment, the assay binding agent is an antigen specific to an antibody to be detected in a sample. antibody specific for a relevant antigen.
11 DK 2005 00290 U4 "Control area", "control strip" and "control line" are used in the same meaning and refer to an area of the membrane to which a control binder is applied.
"Control binder" is any molecule or compound capable of binding specifically. the conjugate or to a control conjugate.
"Test strip" refers to a complete apparatus which can be used to detect the presence of an analyte in a sample. The test strip preferably comprises at least one test membrane having a test region and a control region. As described above, the test strip may be present in a housing such as a Alternatively, the test strip can be used without a housing. If it is in a housing, the test strip and the housing may collectively be termed a "test card".
"Test card window" and "sample window" denote a hole in the plastic housing of a test card through which a sample can be applied. The test card window is preferably above the test area and control area. The test card window also preferably allows a visualization of the test results.
"Buffer window" refers to a hole in a plastic housing on a test board through which buffer can be applied. According to one embodiment, the buffer window is preferably such that it is possible to apply buffer to a buffer pad. In another embodiment, the buffer window is such that it allows application of buffer directly to the test membrane.
"Volume indicator window" refers to an opening in the plastic housing of a test card through which a visible sample volume indicator can be seen. The volume indicator window is located downstream of the sample window.
The sample volume indicator signals that an acceptable amount of sample volume has come into contact with the test area. A 12 DK 2005 00290 U4 sample volume indicator may e.g. may be methylene blue or other colored compound which has been applied to the membrane just upstream of the volume indicator window. The sample volume indicator is carried across the volume indicator window upon movement of the sample, thereby producing a visual signal in the window. In other embodiments, the sample volume indicator is a compound that changes color when brought into contact with the sample, such as a pH indicator. Typically, a sample volume indicator is used when a large sample volume is needed, such as when a relevant analyte is believed to be present at a low concentration in a sample. For example, a sample volume indicator is preferably used when urine is analyzed for the presence of hCG.
"Sample" refers to any material to be analyzed for the presence of a relevant analyte. The sample is preferably in liquid form. Examples of samples include, but are not limited to, body fluids such as whole blood, plasma, serum, saliva and urine. As will be described in more detail below, the sample may be a solid. In this case, however, the sample is preferably made soluble or extracted prior to use in the test.
"Sample application area is the location of the membrane to which sample is applied. The sample application area is preferably downstream of the conjugate source area and upstream of the test area. More preferably, the sample application area comprises the test area. that is in contact with the membrane.
The term "antibody is used in its broadest sense and includes as non-limiting example whole antibodies as well as single-chain antibodies, antibody fragments and chimeric antibodies, provided they have retained the desired binding specificity.
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test card
The diagnostic devices described herein preferably comprise a test card 1, as shown in FIG. 1-3. In FIG. 1 shows a test card 1 comprising a plastic housing 10, with an upper portion 15 and a lower portion 18, as the snapper joint. There are two openings within the upper portion 15 of the plastic housing 10: A sample window 20 and a buffer window 30. The sample window 20 allows application of a sample to the sample application area of a membrane located within the plastic housing 10. The sample application area preferably comprises a test area 140. A control region 150 is preferably located downstream of the test region 140 on the membrane. Buffer window 30 allows application of buffer to conjugate source area 50. The membrane is part of a test strip, which will be described in detail below.
An additional optional volume indicator window (not shown) is located downstream of the test area in some embodiments. The volume indicator window, if present, will allow the user to see an indicator, such as a color change, on the membrane downstream of the test area. The indicator signals that a sufficient amount of sample has contacted the test area and that the user can continue to contact the test area with the conjugate in order to detect bound analyte as described below, e.g. by applying buffer to the conjugate source region.
In FIG. 2, a top view of the test card 1 is shown showing sample window 20 and buffer window 30 within the plastic housing 10. The test area of membrane 140 is accessible through sample window 20 and conjugate source area 50 is available through buffer window 30. Downstream of test area 140, a control area 150 is disposed. Although the windows are shown with a sample window 40 which is rectangular and a buffer window 30 which is circular, they may be of any shape or size. However, they are preferably designed and sized to direct the sample to the sample application area of the membrane 40 and the buffer to the conjugate source region 50. In addition, the sample window is designed to allow the visualization of the test region 140 if a positive result is obtained and the control region 150.
Figure 3 illustrates an example of a test card 1, viewed from the side. The upper part s 15 and the lower part 18 of the plastic housing 10 are shown. The enlarged end (60) of the upper portion 15 of the plastic housing 10 distal to the buffer window (not shown) permits the placement of absorbent material such as an absorbent pad or a desiccant tablet within the test card (1). The size of the enlarged end (60) will vary according to the amount of absorbent material used, which in turn will depend on the volume of the sample and the buffer used in the test.
The test strip
A test strip comprises a test membrane with at least one test area. An analyte binder specific to a relevant analyte is otherwise bound or immobilized at the test site. At least part of the test area becomes visible after completion of the test if the analyte concerned is present in the sample. In a particular embodiment, the test region comprises immobilized antigen and the test allows the identification of particular antibodies in a sample such as a biological fluid. According to a further embodiment, the test area comprises bound antibody and the test allows identification of a particular antigen in a sample.
The sample is applied to a sample application area located downstream of the conjugate source region, e.g. downstream of the conjugate pad. In the preferred embodiment, the sample application area comprises the test area. However, in other embodiments, the sample application area is located upstream of the test area and the sample flows from the sample application area to the test area.
A preferred embodiment of the test strip is shown in diagram form in FIG. A test strip 100 for testing a sample for the presence of a relevant analyte, such as an antibody, preferably comprises a buffer pad 110 in contact with a conjugate pad 120. In contrast, the conjugate pad 120 is in contact with a test membrane 130 comprising a test region 140 and a control region 150. The membrane 130, in turn, is in contact with an absorbent pad 160. Absorbent pad 160 is optionally in contact with an additional absorbent paper 170. Absorbent pad 160 and / or optional absorbent paper 170 may be in further contact with an optional desiccant. 180, such as a desiccant tablet.
In some embodiments, a removable filter holder 190 is attached to the test strip 100. The filter holder 190 comprises a filter paper 200. When the filter holder 190 is attached to the test strip 100, the filter paper 200 is directly above the sample application area, preferably comprising the test region 140 and the control region 150.
Sufficient sample must be provided in the test area to ensure that a visible positive reading is obtained after completion of the test if the analyte concerned is present in the sample. Thus, assays that are suspected to be present in low concentration in the sample, for example antigens such as hCG in urine, require a larger sample volume. Test strip 100 may be designed to accept such larger sample volumes. An example of such an embodiment is shown in FIG. 7, wherein a sample beaker 500 is attached to the test strip 100. The sample beaker 500 comprises a container for holding a plurality of fluid sample. Typically, the sample cup 500 will have an opening at the bottom which allows the sample to flow out of the sample cup 500 and come into contact with the test membrane 130. The sample cup 500 is arranged such that sample flows from the sample cup 500 to the sample application area of the test membrane 130 and Preferably, the sample application area comprises the test region 140 and the control region 150, and the sample beaker 500 is directly above the test region 140 and the control region 150. The sample beaker 500 may optionally comprise a filter through which the sample passes before contacting the test membrane.
The sample may be placed in the sample cup while attached to the test card. In another embodiment, the sample is placed in the test beaker and the test beaker is subsequently attached to the test card and the sample can then flow to the sample application area.
It is desirable to ensure that a sample volume comprising a detectable amount of any analyte present may come into contact with test region 140 before causing the conjugate to flow to test region 140. A sample volume indicator such as a downstream indicator color relative to the test region 140 and a control region 150 can be used to determine if a sufficient amount of the sample has come into contact with the test region 140 and the control region 150. For example. For example, an indicator color such as methylene blue can be applied to absorbent pad 160 or absorbent paper 170 downstream of the test membrane. The flow of liquid sample along absorbent pad 160 or absorbent paper causes the color to flow thereby creating a visual signal, e.g. a visible blue color. The signal can be seen through a volume indicator window in a window in the test board (not shown).
According to some embodiments, especially those where a larger sample volume (greater than about 50 µΙ) is to be applied, the conjugate pad 120 is not in flow communication with the test membrane 130. Such an embodiment is shown in FIG. 7. Separation of Conjugate Pad 120 and 17 DK 2005 00290 U4 Test Membrane 130 prevents conjugate and sample mixing before the sample contacts test area 140. After contacting the test with test region 140 and control region 150, the conjugate pad 120 is moved to comes into fluid contact with the test membrane 130. Then buffer is applied to buffer pad 110 to initiate the test. Buffer can be applied to buffer pad 110 directly through a buffer window by breaking an ampoule 300 where the buffer is present, causing the buffer to flow to buffer pad 110. According to one embodiment, buffer is applied to buffer pad 110 before conjugate pad 120 is brought into flow. According to alternative embodiments, the buffer is applied directly to the conjugate pad 120.
The method by which conjugate pad 120 is brought into fluid communication with membrane 130 is not limited in any respect. According to one embodiment, the user displaces a movable portion of the housing to which conjugate pad 120 and optionally buffer pad 110 is attached, thus contacting conjugate pad 120 with membrane 130. The mechanism for moving conjugate pad 120 in fluid communication with test membrane 110 also preferably receives buffer pack 300 to be broken. The mechanism may also be used to remove sample beaker 500 from test strip 110.
A tablet of desiccant 180 is preferably in fluid communication with the absorbent paper 170. The size of the desiccant tablet 180 is selected after being able to absorb the total sample volume applied to the test membrane 130. The desiccant tablet and any other absorbent material are downstream. for the sample application area and facilitates sample flow across the test area.
According to other embodiments, the sample cup is replaced by a wick brought into contact with a liquid sample. The sample flows along the wall to the test membrane and comes into contact with the test area and the control area. According to a particular embodiment, the wick is placed in the urine stream to analyze the urine for the presence of an analyte such as hCG. In these embodiments, the conjugate pad is preferably not in flow communication with the test membrane during the period when the sample is applied to the wall. After a sufficient amount of sample has come into contact with the test area, the conjugate pad is moved to contact the test membrane. The mechanism for moving the conjugate pad may also have the function of removing the wall. Buffer is subsequently applied to the buffer pad through a buffer window as described above. Alternatively, the buffer located within an ampoule within the test card may be caused to flow to the buffer pad and / or the conjugate pad by breaking or otherwise opening the ampoule.
The test strip is preferably inside a housing, as shown in FIG. 2 and as described above.
In FIG. Figure 5 shows a positive test result indicating the presence of the relevant analyte in a sample. In addition, the test region 150 is visible as a line across the sample window 20 indicating that the conjugate has flowed from the conjugate source region 50 and over the test region 140. In contrast, a negative region is shown. result in FIG. 6. With a negative result, only the control region 150 is visible as a line across the sample window 20. Again, the control indicates that the conjugate has flowed from the conjugate source region 50 and over the test region 140. Thus, the absence of a line in the test region 140 means absence of bottom relevant analyte.
Use of the test strip
A sample to be tested for the presence of a particular analyte which is of interest is applied to the sample application area of the test membrane so that it comes into contact with the test area. According to the preferred embodiment, the sample is applied to the membrane and brought into contact with the test area before the conjugate contacts the test area. Thus, the relevant analyte, if present in the sample, will have the opportunity to bind to the analyte binder in the test region prior to conjugate binding.
If the test strip is in a housing, the sample is preferably applied through a sample window. In that case, the membrane comprises a sample application area which is accessed through the sample window. Preferably, the sample application area comprises at least one positive test area. More preferably, the sample application area comprises a test area and a control area. Alternatively, the sample may be applied by means of a wick or beaker to flow into the sample application area of the membrane and contact the test and control areas.
If the test strip is not in a housing, the sample may be applied directly to the membrane application area of the membrane, preferably including the test area and the control area. Alternatively, the sample may be applied to the membrane in a sample application area upstream of the test area and caused to flow across the membrane to contact the test area. However, the sample is preferably contacted with the test region prior to addition of the conjugate to the membrane.
According to one embodiment, the sample is collected or placed in a sample beaker. The beaker attaches to the housing and allows the sample to flow to the membrane sample application area and come into contact with the test area and control area. A sample cup is preferably used if a sample volume greater than approx. 10μΙ. The sample cup may optionally comprise a filter through which the sample passes before contacting the membrane.
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According to a further embodiment, the sample is applied to a wick which is in contact with the membrane. The wall allows the sample to reach the membrane sample application area and come into contact with the test and control areas. The wall preferably consists of glass paper, polyester, cellulose or other material which will allow the sample to migrate to the membrane comprising the test area. If the test card is in a housing, the wall may protrude relative to the housing, thereby allowing access to the wall for application of the sample. For example, if the presence of a particular analyte in urine, e.g. hCG, the wick can be placed in the urine stream by the subject to be tested. According to a further embodiment, the wick can be contacted with the sample to be analyzed, e.g. body fluids such as saliva.
The sample to be tested is preferably applied to the sample application area of the membrane in liquid form. Examples of samples that can be analyzed include, but are not limited to, serum, plasma, whole blood, saliva and urine. Fully or semi-solid specimens can be dissolved and applied to the membrane or extra cured before application.
According to other embodiments, sample buffer, which preferably does not contain conjugate, is applied to the membrane prior to application of the sample. In some embodiments, the sample buffer comprises a blocking agent. Preferably, the sample buffer is applied to the membrane in the sample application area. From approx. 1 to approx. Preferably, 25 μΙ of sample buffer is applied to the membrane, more preferably from ca. 1 to approx. 10 μΙ, further preferably approx. 1 to 5 μΙ. According to a particular embodiment, 5 μΙ of sample buffer is applied to the membrane sample application area, followed by 5 μΙ of serum, plasma, whole blood or other liquid sample. Application of sample buffer prior to application of the wash buffer is especially preferred when testing for HCV as described below and when using viscous samples. Using 21 DK 2005 00290 U4 sample buffer increases the sensitivity of the test and viscous samples flow faster.
Sample buffer preferably comprises a buffer agent and a preservative. It may also include one or more detergents and / or sugars. Any buffering agent may be used, preferably in an amount which maintains the desired pH of the test buffer. Preferably, the pH of the sample buffer is from about 7.2 to 7.6. For example, and without limitation, the buffer may be selected from the group consisting of HEPES, Tris and phosphate buffers. According to a preferred embodiment, the sample buffer comprises from ca. 0.01% to approx. 0.5% HEPES, more preferably from ca. 0.02 to approx. 0.05% HEPES. According to a further embodiment, the sample buffer comprises from ca. 0.01% to approx. 0.5% phosphate, more preferably from ca. 0.02 to approx. 0.05% phosphate. One skilled in the art will be able to adjust the amount of buffer to maintain the desired pH.
The sample buffer also preferably comprises a preservative such as sodium azide or Proclin. According to a preferred embodiment, the sample buffer comprises from ca. 0.1% to approx. 1.0% sodium azide, more preferably from ca. 0.1% to approx. 0.2%, further preferably approx. 0.2%.
Depending on the nature of the sample to be tested, the sample is optionally applied to the membrane through a filter. Examples of sample materials preferably filtered include whole blood, stool samples, urine, food extracts and soil extracts. According to one embodiment, whole blood is applied through a membrane filter. The filter removes contaminants and allows analyzers, such as antibodies, to pass, thereby increasing the sensitivity of the test. The filter is preferably enclosed within a holder which can be attached to the test card or test strip as needed. If attached to a test card, the filter is preferably attached directly above the sample window. The filter medium is selected based on the type of sample to be filtered. Such a choice lies within the competences of a professional. For example, the filter material for whole blood samples may be, but is not limited to, the Pall BTS-SP300®.
As discussed above, the filter is preferably attached to the test strip with a removable filter holder, although the filter for special applications could be permanently attached. The filter holder is typically a device that snaps into the housing of the test card. The design and type of the device may vary, but it is typically made of polystyrene, polyethylene, polypropylene or other plastic material.
The test region is a portion of the membrane covered with a binder, preferably a protein capable of binding specifically to the relevant assay. For example, the positive test region may be coated with antigen specific to a relevant antigen. According to particular embodiments, the test area comprises bound HIV antibody or hepatitis antibody, as described in more detail in the examples below. When the test area comprises antigen, preferably from 0.025 to approx. 0.4 µg antigen.
In other embodiments, an antibody specific for a relevant antigen is immobilized in the assay region. The antibody may be a polyclonal or monoclonal antibody. When the test area comprises antibody, preferably from 0.1 to approx. 20 µg antibody, more preferably from ca. 0.2 to approx. 2 pg, further preferably from about 0.5 to approx. 1 pg. According to a particular embodiment, the test region comprises an antibody to hCG, preferably a monoclonal antibody to hCG.
The binder can be applied to the membrane in any desired pattern and it follows that the visible result of a positive test can have any shape. According to a preferred embodiment, the test area is linear and forms a line extending from one side of the membrane to the other, so that a vertical line appears across the sample window, if a positive result is obtained, an analyte duct in the sample is indicated. According to other embodiments, the test area is designed as a symbol such as one
It is possible to test for more than one type of analyzer with a single test strip. In that case, additional positive test areas are provided on the test membrane. For example, another positive assay region may include antigen against another type of antibody or antibody to another type of antigen. The binder at a further positive test range need not be of the same type as the binder at a first test range. Thus, the binder at a first test region may be an antibody, while a binder at a second test region may be an antigen. In this way, a sample can be analyzed for occurrence of more than one type of analyte.
Each additional positive test area may have a different shape from the first test area to make it easy for the user to distinguish between the nature of the positive results. Depending on the type of additional analyte to be tested for, additional conjugates may be required that bind the different analyte. For example, if the relevant analyzers. are two different antibodies, a single conjugate comprising protein A. may be used. binds the antigen.
The control region preferably comprises a control binder which will bind to the conjugate. By positioning the control region downstream of the positive test region, a reaction at the control area would indicate that the conjugate migrated past the test area where available and could respond if the relevant analyte was present. If the conjugate is designed to recognize an antibody, the control region preferably comprises an immunoglobulin.
H
24 DK 2005 00290 U4 such as rabbit IgG. If the conjugate comprises an antibody, the control region preferably comprises an antibody binder such as protein A. The control region may be of any desired form, such as a line or symbol, e.g.
5
According to other embodiments, the control region comprises a control binder which binds to a control conjugate. For example, the control region may comprise β-gal and the control conjugate may comprise β-galactosidase conjugated to gold.
The binder and control binder may be applied to the test membrane by any method known in the art. They are typically applied by contacting the test membrane with the binder such as by spraying or contacting the test membrane using a pump metering system. Such systems are commercially available e.g. from Kinematic Automation.
When the sample is in contact with the contact area, the relevant analyte, if present in the sample, will bind to the binder at the test site. Excess unbonded analyte is washed away from the test region by buffer migration along the membrane as described below.
After the sample is applied to the membrane, the wash buffer is applied to the test membrane. Preferably from ca. 1 to approx. 20, more preferably from ca. 1 to about 5 drops of wash buffer are initially applied. Additional wash buffer may subsequently be applied if needed to cope with the reaction.
The wash buffer preferably carries the conjugate to the test region. Wash buffer can be applied directly to the test membrane. However, according to the preferred embodiment, wash buffer will be applied to an upstream buffer pad from which it flows to the test membrane. The buffer pad can be made of any material that may contain the buffer and allows it to flow through to the membrane at the desired rate. For example, the buffer pad may be one or more materials selected from the group consisting of glass paper, cellulose and polyester. According to a preferred embodiment, the buffer pad is a Whatman GF / DVA glass paper. Those skilled in the art will be able to select a suitable material for the buffer pad on the basis of factors such as the design of the housing in which the test strip with lateral flow is located, the desired flow rate, and the amount of buffer to contain the buffer pad.
The wash buffer can be applied directly to the buffer pad, e.g. with a pipette or drip counter. However, according to another embodiment, the appropriate amount of wash buffer is in a container adjacent to the buffer pad. For example, the wash buffer may be in a glass or plastic cartridge. Upon application of the test sample, the container containing the buffer is broken or otherwise opened allowing the wash buffer to come into contact with the buffer pad.
The composition of the wash buffer can be varied to increase the sensitivity of the test. Preferably, the wash buffer comprises a buffering agent, a preservative, a detergent, sugar as well as other materials which reduce background and / or nonspecific binding.
Any buffering agent may be used provided it maintains the desired pH of the buffer, preferably from ca. 7.2 to approx. 7.6. For example and non-limiting, the buffer may be selected from the group consisting of HEPES, Tris and phosphate buffer. According to a preferred embodiment, the wash buffer comprises from ca. 0.01% to approx. 0.5% HEPES, more preferably from ca. 0.02 to approx. 0.2% HEPES, and further preferably from ca. 0.02 to approx. 0.05% HEPES. According to a particularly preferred embodiment, the wash buffer comprises 0.025% HEPES, 26 DK 2005 00290 U4
The wash buffer may also comprise a preservative such as sodium azide or Proclin. According to a preferred embodiment, the wash buffer comprises from ca. 0.1 to approx. 0.5% sodium azide, more preferably approx. 0.1 to approx. 0.2%.
Sodium chloride or a comparable salt is preferably present at a concentration of ca. 1%, more preferably approx. 0.85%. The EDTA may be present preferably at a concentration of ca. 0.1%. A sugar such as mannitol may also be present. Preferred mannitol concentrations range from approx. 1% to approx. 5%. According to a preferred embodiment, the mannitol concentration is approx. 1%.
Preferably, the wash buffer also contains a blocking agent. The blocking agent may be any blocking agent known in the art. Preferably, the blocking agent is a protein such as casein or bovine serum albumin (BSA). The blocking agent is preferably present in the wash buffer at a concentration of from ca. 0.01 to approx. 0.5%, more preferably from ca. 0.02 to approx. 0.2%. In a particularly preferred embodiment, casein is present in the wash buffer at a concentration of ca. 0.1%.
The wash buffer typically also includes a detergent, preferably a nonionic detergent such as Triton X100 ™ or Tween. Preferably, the detergent is present at a concentration of ca. 0.05 to approx. 1%, more preferably from ca. 0.1 to approx. 0.5%. According to a particularly preferred embodiment, the buffer comprised Triton X100 at a concentration of approx. 0.1%.
The pH of the wash buffer was adjusted to between ca. 7 and 8, more preferably to between ca. 7.2 and 7.6. According to a particularly preferred embodiment, the pH of the wash buffer is about 7.2.
The test strip also includes a conjugate source region. According to a preferred embodiment, the test strip comprises a conjugate pad on which the conjugate is dried as described below. Wash buffer flows from the buffer pad to the conjugate pad with which it is in contact. The conjugate pad, in turn, is in contact with the test membrane. However, in some embodiments, the conjugate pad is contacted with the membrane after application of the sample. Especially when a large sample volume is used, the conjugate pad is preferably brought into contact with the membrane after the sample has come into contact with the test area. ·.
Sufficient wash buffer is applied to the buffer pad to dissolve the conjugate which is diffusion bound to the conjugate pad and cause the conjugate to flow along the test membrane to the test line and control line. The amount of buffer to be used may be determined by one of ordinary skill in the art on the basis of certain factors such as the amount of conjugate bound to the conjugate pad, the size of the membrane, and the amount of sample to be analyzed. Preferably, from 1 to 10 drops of buffer solution is applied to the buffer pad when the buffer pad is approx. 2 cm x 5 mm, more preferably from 2 to 5 drops, further preferably 3 to 4 drops.
According to alternative embodiments, no conjugate pad is provided. According to one alternative embodiment, the conjugate pad is present in the wash buffer at a concentration ranging from ca. 0.2 OD to approx. 1.0 OD / ml. The wash buffer containing the conjugate may be applied to the buffer pad. The conjugate thus flows with the wash buffer from the buffer pad to the membrane, where it contacts the test area and the control area. According to another embodiment, wash buffer containing conjugate will be applied directly to the test membrane. Washing buffer containing the conjugate can be provided in an ampoule and caused to flow to the membrane by breaking or otherwise opening the ampoule. The wash buffer containing the conjugate is preferably applied to the membrane at a point upstream of the test region. However, in some embodiments, the wash buffer comprising the conjugate will be applied directly to the test area after the sample has been applied.
According to a further embodiment, the conjugate, such as by drying, is diffusely bonded directly to the membrane comprising the test region and the control region. Wash buffer can then be applied to a buffer pad which is in contact with the membrane or directly on the test membrane. The wash buffer dissolves the dried conjugate and carries it across the test membrane to the test area and control area.
The wash buffer containing conjugate preferably contacts the test area after application of the sample to any analyte present in the sample having the opportunity to bind the immobilized binder. Thus, the wash buffer is preferably applied to the test strip after the sample is applied to the test area.
The conjugate described above in detail is prepared for use in the test by dilution in conjugate buffer. Preferably, the working conjugate is prepared by diluting the conjugate in the conjugate buffer to ca. 3 OD / mL. The composition of the conjugate buffer can be varied according to the nature of the conjugate. Examples of conjugate buffer include a buffer such as HEPES, Tris, phosphate or other similar buffer known in the art. A preferred conjugate buffer comprises between ca. 0.02 and 0.2% HEPES buffer, more preferably ca. 0.025% HEPES. The conjugate buffer may also comprise a salt, preferably sodium chloride. Sodium chloride is added until a concentration of approx. 0.1%, more preferably 0.85%. A preservative may also be added to the conjugate buffer, such as a sodium azide or Proclin, at a concentration of between ca. 0.1 and 0.2%.
The conjugate buffer also preferably comprises EDTA (about 0.1%), casein at a concentration of between about 0.02 and approx. 0.2%, more preferably approx. 0.15%, 29 bovine serum albumin (BSA) at a concentration of between 0.5% and approx. 3%, more preferably approx. 1%, mannitol at a concentration of between ca. 1% to approx. 5%, more preferably approx. 1%, sucrose at a concentration of between approx. 1% and approx. 5%, more preferably approx. 1%. The Triton X100 may be included in the buffer, preferably at a concentration of between ca. 0.1 and approx. 0.5%, more preferably approx. 0.1%.
Preferably, the conjugate buffer is adjusted to a pH of between ca. 7.2 and approx. 7.6, more preferably to a pH of ca. 7.2.
According to one embodiment, after preparing the working conjugate by diluting the conjugate in the conjugate buffer, the working conjugate is dried on a conjugate pad, e.g. by vacuum drying. The conjugate pad is preferably cut into an appropriate size strip for use in conjunction with the test strip. In the examples below, the conjugate pad is cut into strips of 5 mm x 5 mm.
The conjugate pad preferably comprises glass fibers, glass paper, polyester or cellulose paper. These materials are well known in the art and are e.g. available from Millipore, S&S and Whatman. According to a preferred embodiment, the conjugate pad is a commercially available fiberglass conjugate pad GFCP203000 (Millipore).
As described above, the wash buffer dissolves the dried conjugate and causes it to flow from the conjugate pad and along the test membrane past the test area and control area.
The test membrane may be any material to which a protein or other binder may be attached, either co-valent or non-co-valent, and the conjugate may flow with the wash buffer along it. The test membrane is preferably a nitrocellulose membrane. Other membranes with higher or lower flow rates may be selected for use as a test membrane by those skilled in the art on the basis of factors such as e.g. the desired sensitivity and test times and costs.
As the conjugate flows with the wash buffer across the membrane, it comes into contact with the test area. If the relevant analyte is present and binds to the binder at the test site, 1 conjugate in the buffer will bind to the analyte.
The wash buffer comprising conjugate which was not bound at the test region will continue to flow across the membrane to the control region. Conjugate will bind to the control binder. The wash buffer will also carry unbound sample from the test area and control area.
Washing buffer comprising unbonded conjugate and unbonded sample will then flow to an absorbent pad which is in contact with the downstream end of the membrane. The absorbent pad can be made of any absorbent material known in the art. Preferably, the absorbent pad is an absorbent paper such as S&S 900 ™ paper. Other types of absorbent paper are well known in the art and may be selected by one of ordinary skill in the art depending on the final design of the test strip.
The wash buffer may continue to flow through the absorbent pad and into an absorbent paper which is in contact with the absorbent pad. The absorbent paper is preferably used if a large amount of buffer is to be absorbed.
A molecular desiccant tablet may also be incorporated into the test strip. In one embodiment, the buffer with unbound conjugate flows from the absorbent paper into the desiccant tablet with which the paper is in contact. The desiccant tablet retains the absorbed fluid and prevents backflow into the test strip.
The desiccant tablet can also keep the test strip dry until use. A preferred dry strap delta blend is a 0.395g Tri-Sorb tablet, available from Sud-Chemie (product no. 43-01).
The person skilled in the art can choose the appropriate size and material for the absorbent pad, absorbent paper and desiccant tablet according to the final design of the test strip, including the total volume of sample and buffer to be used.
The entire test strip is preferably inside a housing, e.g. a plastic housing. As described above, the housing according to one embodiment comprises a sample window where through the sample the membrane containing the test and control areas can be applied. The housing may also comprise a buffer window through which buffer buffer or diaphragm may be applied. The housing may also include a volume indicator window through which a signal can be read indicating that a sufficient amount of the sample has come into contact with the test area. A sufficient amount of the sample is a sample amount that is considered to contain enough of the relevant analyte to, if present, that a visible signal will be generated at the test area after completion of the test if the analyte is present in the sample. For example, if a test. should be able to detect hCG in urine, a sufficient sample volume will be approx. 200 μΙ urine.
The test results are preferably read between approx. 1 and 5 minutes after adding wash buffer to the test strip. According to one embodiment, the results from ca. 2 to 4 minutes after the addition of wash buffer, more preferably from ca. 3 to 4 minutes after adding wash buffer. According to a particularly preferred embodiment, the results are read 1 minute after the addition of wash buffer. As shown in FIG. 7, the test line (300) will be visible as a positive result. A visible control strip (400) indicates that the test was working properly. As shown in FIG. 6, a negative result is indicated by the control line (400) being visible while the test line is not.
Example 1: The construction of an HIV test card
A test with lateral flow and rapid visual display was made to detect antibodies to human immunodeficiency virus. The test strips were constructed essentially as shown in FIG. First
The test strips included a plastic backing (6 cm x 5 cm) with adhesive on both sides. The plastic backing was between approx. 2 μιτι and approx. 20 μιτι in thickness.
A test line and a control line were prepared on nitrocellulose membrane (Millipore HF09004) by spraying with a pump dosing system (Kinematic Automation). The test line included a vertical line of HIV antigen. The HIV antigen was a mixture of HIV-1 recombinant glycoprotein antigens (GP120 (about 0.15 mg / ml) and P24 (about 0.1 mg / ml)), and HIV-2 recombinant glyco-protein antigen (GP36 (about 0.5 mg / ml) at a concentration of from about 0.5 to about 1.0 mg / ml prepared in PBS comprising 5% trehalose. 0.5 µg of antigen was applied to the test area in a vertical line.
Other test strips were prepared using a single HIV-1 or HIV-2 antigen on the test line.
For all test strips, the control area included a vertical line of control antibody. The control antibody was rabbit IgG diluted to 1 mg / mL in PBS comprising 5% Trehalose. A total amount of control antigen sufficient to produce a visible control line after conjugate binding was applied to the control region.
33 DK 2005 00290 U4
For each test strip, appropriate nitrocellulose membrane was cut to a size of approx. 2.5 cm x 5 mm and attached to the plastic backing.
Protein A attached to colloidal gold marketed by Sigma was used as a conjugate for the test. The conjugate was diluted in conjugate buffer to a concentration of ca. 3 OD / ml. The conjugate buffer comprised 0.25% HEPES; 0.85% sodium chloride, 0.1% sodium azide, 0.1% EDTA, 0.1% casein, 1% bovine serum albumin, 1% mannitol, 5% sucrose and 0.1% Triton X100. The buffer was adjusted to a pH of 7.2.
The conjugate was applied to a fiberglass conjugate pad (Millipore GFCP203000) which was dried and cut into 5 mm x 5 mm strips. A strip was attached to the end of the nitrocellulose membrane closest to the test area of each test strip.
A Whatman GF / DVA glass paper buffer pad (2 cm x 5 mm) was then attached to the dried conjugate pad of each test strip to serve as a buffer pad.
An absorbent pad (2 cm x 5 mm) made of S&S 900 paper was attached to the end of the nitrocellulose membrane closest to the control area and an additional absorbent paper (2.5 cm x 5 mm, S&S 470) was attached to the absorbent pad. In contrast, the absorbent paper was contacted with a 0.395 g desiccant tablet (Tri-Sorb, Sud-Chemie).
The test strip is placed in a plastic housing to produce a test card similar to that of FIG. 1-6. The plastic housing (10) included a test window (20) which exposed the test area (140) and the control area (150), as well as a buffer window (30) directly above a buffer pad. The plastic housing is also provided with a marking to indicate the location of the test line and the control line.
34 DK 2005 00290 U4
The test cards were individually wrapped in foil.
Example 2: Test for HIV
Serum or plasma was prepared from a whole blood sample obtained from a patient to be tested for HIV using proper venipuncture techniques. The samples were stored at between 2 ° C and 8 ° C and used within 24 hours or frozen at -20 ° C for use within two weeks. Frozen samples thawed before use.
An HIV test card as described in Example 1, comprising a mixture of HIV-1 and HIV-2 antigens in the control area, was removed from the foil package.
Ca. 5 µ! sample buffer comprising 0.025% HEPES and 0.2% sodium azide was applied to the membrane through the center of the test card window.
A sample pipette containing the serum or plasma to be examined is held in a vertical position above the test window on the test card and a 5 μΙ sample is placed on the membrane in the middle of the window.
Four drops of wash buffer solution (0.025% HEPES, 0.85% sodium chloride, 0.1% EDTA, 1% mannitol, 0.1% casein, 1% Triton X100, pH 7.2) were added to the buffer. After three minutes, one more drop of buffer was added to the buffer pad if the reaction had not become clear.
The results of the test are read from 1 to 5 minutes after the addition of the first buffer.
As will be seen from FIG. 5, a positive result comprises two visible lines across the sample window: a test line (140) and a control line (150). A positive is obtained if two lines are visible, even if one of the lines is darker than the one. other. A negative result is indicated by the fact that a single control line is visible across the window, as will be seen in FIG. 6th
Example 3: HCV test card
A test strip for the diagnosis of hepatitis C virus was prepared in the same manner as in Example 1. However, a hepatitis C antigen mixture comprising core (0.3 mm / ml), NS3 (0.4 mg / ml), and NS4 (0) was sprayed. , 1 mg / ml) in a membrane test line to produce the test site in place of HIV antigen. A total of approx. 0.4 µg of HCV antigen mixture was applied to the test line. Rabbit IgG diluted to 1 mg / ml was sprayed in a control line.
Example 4: Test for HCV
An HCV test card as described in Example 3 comprising an HCV antigen in the test area was removed from its foil wrapper,
Ca. 5 µΙ sample buffer comprising phosphate buffer and 0.2% sodium azide was applied to the membrane through the center of the test card window.
Serum or plasma was generated from a whole blood sample. A sample pipette containing serum or plasma to be tested is held vertically above the test window on the test card, and a 5 μΙ sample is placed on the membrane in the center of the window.
Four drops of wash buffer solution (0.025% phosphate, 0.85% sodium chloride, 0.1% EDTA, 1% mannitol, 0.1% casein, 1% Triton X100, pH 7.2) were added to the buffer. After 3 minutes, a further 2 drops of buffer were added to the buffer pad if the reaction was not completed.
36 DK 2005 00290 U4
The result of the test is read 1 to 5 minutes after the addition of the first buffer.
As shown in FIG. 5, a positive result comprises two visible lines across the sample window: a test line (140) and a control line (150). A positive result is achieved if two lines are visible, even if one line is darker than the other. A negative result is shown with a single control line visible across the sample window as shown in FIG. 6th
Example 5: Pregnancy test
A rapid visual pregnancy test with lateral flow was prepared. The arrangement of the test strips is substantially as shown in FIG. 7th
Each test strip includes a plastic backing (6 cm x 5 mm) with adhesive on both sides. The plastic backing is between approx. 2 pm and approx. 20 µm in thickness.
A test line and a control line were prepared on a nitrocellulose membrane (Millipore HF09004) by spraying with a pump dosing system (Kinematic Automation). The test line comprises a vertical line of an antibody to human chorionic gonadotropin (hCG), preferably a monoclonal antibody. Antibodies to hCG are widely available commercially, e.g. from Research Diagnostics Inc. (RDI-CBL74; New Jersey, USA), Charles River Labs or other sources or may be manufactured in accordance with standard procedures. A total amount of from approx. 0.2 to approx. 1 µg antibody is applied to the test area in a vertical line.
The control line comprises a vertical line of Protein A. A total amount of protein A sufficient to produce a visible signal after conjugate binding is applied to the control line, typically approx. 0.25 pg.
37 DK 2005 00290 U4
For each test strip, the appropriate nitrocellulose membrane is cut to a size of approx. 2.5 cm x 5 mm and attached to the plastic backing.
5
Another antibody to hCG is attached to colloidal gold to the conjugate of the assay. Colloidal gold hCG antibody conjugates are commercially available e.g. from Research Diagnostics Inc. Alternatively, hCG antibody conjugates may be prepared by well known methods (see, e.g., U.S. Patent No. 10,485,982, incorporated herein by reference). The conjugate is diluted in conjugate buffer to a concentration of ca. 3 OD / ml. The conjugate buffer preferably comprises 0.25% HEPES, 0.85% sodium chloride, 0.1% sodium azide, 0.1% EDTA, 0.1% casein, 1% bovine serum albumin, 1% mannitol, 5% sucrose, and 0.1% Triton X100. The buffer is adjusted to a pH of 7.2.
The conjugate is applied to a fiberglass conjugate pad (Millipore GFCP203000) which was dried and cut into strips of 5 mm x 5 mm.
A Whatman GF / DVA fiberglass buffer pad (2 cm x 5 mm) was attached to the dried conjugate pad on each test strip as a buffer pad.
An absorbent pad (2 cm x 5 cm) made of S&S paper was attached to the end of the nitrocellulose membrane closest to the control area and an additional 25 absorbent paper (2.5 cm x 5 mm; S&S 470) was attached to the absorbent pad. The absorbent paper, in turn, comes into contact with a desiccant tablet (e.g., Tri-Sorb, Sud-Chemie), which is capable of absorbing a substantial amount of the sample volume, thereby drawing sample and analyzer over the test area.
30 38 DK 2005 00290 U4
Methylene blue or other indicator is deposited on either the absorbent pad or absorbent paper downstream of the test area. The movement of this volume indicator is a signal that a sufficient amount of sample has come into contact with the test area for the test to be accurate.
The test strip is placed in a plastic housing to produce a test card. The plastic housing includes a test window above the test area and control area to enable visualization of the test results. The housing also includes a volume indicator window through which the movement of the volume indicator can be observed.
Some test cards attach a sample cup over the test window. The sample cup comprises a container in which the sample is to be stored and includes an outlet so that the sample can flow to the sample application area of the test membrane through the sample window.
The sample cup may also include a filter in the outlet. After adding sample to the membrane, the beaker is removed so that the result of the test can be read.
In other test cards, a wick is provided extending from the end or side of the housing. The wall provides a path through which the sample can flow to the sample application area and test area.
The housing also has a mechanism which allows the conjugate pad to be moved to flow with the diaphragm.
Some test cards also include an ampoule containing wash buffer in the housing upstream of the conjugate pad. For other test cards, buffer is added to the buffer pad through a buffer window.
Activating the mechanism that moves the conjugate pad to contact the test membrane breaks the ampoule, thereby allowing buffer to flow to the conjugate pad. Preferably, approx. 5 drops of wash buffer in contact with the conjugate pad. Activation of the mechanism also removes the sample cup or wick from the housing.
5 The plastic housing is provided with markings to indicate where the test line and control line are in the sample window.
Example 6: Use of Pregnancy Test Pregnancy Test 10 Rapid visual visual pregnancy tests with lateral flow comprising a test cup were prepared as described in Example 5.
Individual urine is collected and approx. 500 µΙ is added to a sample beaker attached to the test card above the sample window. When a blue color is observed in the 15-volume indicator window, the mechanism is activated, whereby the conjugate pad is brought into flow with the membrane. This breaks the vial containing buffer and the test begins. The sample cup is removed and the result of the test can be read in the sample window from 1 to 5 minutes late.
20 As will be apparent from FIG. 5, a positive result comprises two visible lines across the sample window: a test line 140 and a control line 150. A positive result is obtained if two lines are visible, although one of the lines is darker than the other. A negative result is indicated by the fact that there is a single control line 150 visible across the sample window, as shown in FIG. 6th
25
Example 6: Use of a pregnancy test with wick
Rapid visual visual pregnancy tests with lateral flow comprising a wick were prepared as described in Example 5.
30 40 DK 2005 00290 U4
The individual places the wick in his stream of urine. When a blue color can be seen in the volume indicator window, a sufficient amount of sample volume has come into contact with the test area.
A mechanism on the housing is actuated whereby the conjugate pad is brought into fluid flow with the membrane. This also breaks the ampoule containing buffer inside the housing and initiates the test by causing buffer to flow to the conjugate pad. The wall is removed and the test results can be read in the sample window between 1 and 5 minutes later.
As shown in FIG. 5, a positive result comprises two visible lines across the sample window: a test line 140 and a control line 150. A positive result is obtained if two lines are visible, even if one of the lines is darker than the other. A negative result is indicated by a single control line across the sample window, as shown in FIG. 6th
Although the invention is described with reference to given preferred embodiments, it will be appreciated by those skilled in the art that various modifications may be made and equivalents may substitute elements thereof without departing from the scope of the invention. In addition, many modifications can be made to adapt the disclosed herein to a specific situation, without departing from the scope of the invention. Accordingly, it is intended that the invention should not be limited to any specific embodiment described, but that the invention includes all embodiments which fall within the scope of the appended claims.

权利要求:
Claims (36)
[1]
A diagnostic test strip for detecting a relevant analyte in a liquid sample, the test strip comprising: - a conjugate source region comprising a conjugate designed to bind the relevant analyte; a sample application area located downstream of the conjugate source region and configured to receive the liquid sample; and - a test region located downstream of the conjugate source region and comprising immobilized binder specific to the analyte concerned.
[2]
The test strip of claim 1, wherein the sample application area comprises the test area.
[3]
The test strip according to claim 1, wherein the relevant analyte is an antibody.
[4]
The test strip according to claim 3, wherein the relevant assay is an antibody to human immunodeficiency virus (HIV).
[5]
The test strip according to claim 3, wherein the relevant assay is an antibody to hepatitis C virus (HCV).
[6]
The test strip according to claim 1, wherein the relevant assay is an antigen.
[7]
The test strip according to claim 6, wherein the relevant analyte is human chorionic gonadotropin (hCG).
[8]
The test strip of claim 1, wherein the conjugate source region comprises dried conjugate. 42 DK 2005 00290 U4
[9]
The test strip of claim 8, wherein the conjugate source region comprises a conjugate pad comprising dried conjugate and is in flow communication with the test strip.
[10]
The test strip according to claim 8, wherein the conjugate source region is part of the test strip.
[11]
The test strip of claim 1, wherein the conjugate source region is caused to flow to the test region by contacting the conjugate source region with wash buffer.
[12]
The test strip of claim 11, wherein the wash buffer comprises a blocking agent protein and a nonionic detergent.
[13]
The test strip according to claim 12, wherein the wash buffer comprises 0.1% casein and 1% Triton X100.
[14]
The test strip of claim 1, wherein the conjugate comprises a binding component and a label.
[15]
The test strip of claim 14, wherein the binding component is protein A.
[16]
The test strip according to claim 14, wherein the label is selected from the group consisting of colloidal gold, coiloid silver, colloidal black and colored latex particles.
[17]
The test strip of claim 16, wherein the label is colloidal gold.
[18]
The test strip according to claim 17, wherein the colloidal gold has a particle size of from approx. 20 to approx. 80 nm. 43 DK 2005 00290 U4
[19]
The test strip of claim 3, wherein the conjugate comprises protein A conjugated to colloidal gold.
[20]
The test strip of claim 6, wherein the conjugate comprises an antibody conjugated to colloidal gold.
[21]
The test strip of claim 1, wherein the test strip comprises a nitrocellulose membrane.
[22]
The test strip of claim 1, wherein the test strip further comprises a control region located downstream of the test region, said control region comprising an immobilized control binder capable of binding the conjugate.
[23]
The test strip of claim 22, wherein the control region comprises immobilized rabbit IgG.
[24]
The test strip of claim 22, wherein the control region comprises immobilized protein A.
[25]
The test strip of claim 1, further comprising a buffer pad which is in flow communication with the conjugate source region.
[26]
The test strip according to claim 1, further comprising an absorbent pad in flow communication with the membrane at a position downstream of the test area.
[27]
The test strip of claim 1, further comprising a sample filter located directly above the sample application area.
[28]
A test strip for detecting anti-HIV antibody in a sample comprising: 44 DK 2005 00290 U4 - a test region and a conjugate source region located upstream of the test region; immobilized antigen bound at the test site, wherein the immobilized antigen comprises from ca. 0.1 to approx. 0.5 µg HIV antigen; and - a conjugate which is diffusion bound to the conjugate source region, wherein the conjugate is designed to bind an antibody.
[29]
The test strip of claim 28, wherein the HIV antigen comprises one or more antigens selected from the group consisting of GP120, P24 and GP36.
[30]
The test strip of claim 28, wherein the conjugate comprises protein A.
[31]
The test strip of claim 28, wherein protein A is conjugated to colloidal gold.
[32]
An immunodeficiency pregnancy test strip comprising: - a sample application area for receiving a fluid sample; a test area comprising an immobilized first anti-hCG antibody; and - a conjugate source region comprising conjugate designed to bind hCG where the conjugate source region is located upstream of the sample application region and the test region.
[33]
A pregnancy test according to claim 32, further comprising a wick which is in flow communication with the sample application area.
[34]
A pregnancy test according to claim 32, further comprising a beaker for collecting a urine sample.
[35]
The pregnancy test of claim 32, wherein the conjugate comprises a second hCG antibody. DK 2005 00290 U4 45
[36]
The pregnancy test according to claim 35, wherein the second hCG antibody is conjugated to colloidal gold.

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法律状态:
2014-03-28| UUP| Utility model expired|Expiry date: 20140319 |

优先权:

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