RU58717U1 - DIAGNOSTIC TEST STRIP - Google Patents

Abstract

The invention provides diagnostic devices and diagnostic methods. Diagnostic devices preferably comprise a test strip with a test section 140. The sample to be analyzed is in contact with a test section that contains a specific binding partner for the analyte of interest. The analyte, if present in the sample, binds to the immobilized binding partner in the test site and then contacts the conjugate. The conjugate specifically binds the analyte and provides a visual indication of the presence of the analyte. The proposed device can be used to diagnose specific diseases or disorders of the patient, such as HIV or hepatitis. They can also be used to detect pregnancy.

Description

Utility Model Scope

This utility model relates to a quick visual test with a fluid droplet spreading in a radial direction (with side flow) to detect analytes in a sample.

Prior art

Radial spreading test devices are known and can be used in clinical diagnostics to determine the presence of an analyte of interest in a sample, such as body fluids. However, such devices typically require the use of a large sample volume and large quantities of conjugate. They also have a long wait time until the test results can be read.

Utility Model Essence

An immunodiagnostic test strip is proposed for detecting an analyte of interest in a sample. In some embodiments, the analyte is an antibody, such as an anti-human immunodeficiency virus (HIV) antibody or an hepatitis C anti-virus (HCV) antibody. In other embodiments, the analyte is an antigen, such as human chorionic gonadotropin (hCG).

The diagnostic test strip advantageously comprises a conjugate source region that contains a conjugate configured to bind the analyte of interest, a sample application region located downstream of the conjugate source region and configured to produce said liquid sample, and a test section. The test site is located downstream of the conjugate source region and contains an immobilized binder that is capable of

specifically bind to the analyte of interest. For example, if of interest; the analyte is an antibody, the positive test site predominantly contains immobilized antigen. If the analyte of interest is an antigen, then the positive test site predominantly contains an immobilized antibody. The conjugate contains a first binding component that is capable of binding the analyte of interest and a second visualization component.

The sample used in the test can be any liquid. Examples of preferred samples include, for example, blood, serum, plasma, saliva and urine. The sample is applied to a positive test site and then the conjugate is forced to flow out from the area of the conjugate source. The conjugate flows through the membrane and contacts any analyte associated with the positive test site, creating a visual signal.

According to a preferred embodiment, the test strip contains a test site with an immobilized HIV antigen and is used to detect an anti-HIV antibody. In this embodiment, the test is useful for diagnosing a patient's HIV infection. In another preferred embodiment, the test strip contains a test site with an immobilized HCV antigen and is used to detect anti-HCV (HCV = hepatitis C virus) antibodies in the sample. In this embodiment, the test is useful for diagnosing a patient's hepatitis C virus infection. In yet another embodiment, the test strip comprises a test site with immobipized anti-hCG antibodies. In this embodiment, the test is useful for diagnosing the presence of pregnancy.

The conjugate source region preferably contains dried conjugate. For example, the conjugate source region may be a conjugate pad,

on which the conjugate was dried. The conjugate pad predominantly has fluid communication with the membrane. In some embodiments, the conjugate pad is moved and contacted with the membrane after adding the sample to the sample application area. In another embodiment, the conjugate source region is a portion of the membrane on which the conjugate has been dried.

The dried conjugate is predominantly resuspended and induced to flow due to contacting the region of the conjugate source with the wash buffer. In one embodiment, the wash buffer contains about 0.1% blocking agent, preferably a blocking protein, such as casein. The wash buffer also predominantly contains about 0.1% detergent, mainly a non-ionic detergent, such as Triton X100 ^TM . In one embodiment, the wash buffer contains about 0.025% HEPES, about 0.85% sodium chloride, about 0.1% EDTA (ethylenediaminetetraacetate), about 1% mannitol, about 0.1% casein, and about 1% Triton X100 ^TM detergent.

In another embodiment, the conjugate is suspended in a buffer. The conjugate is forced to flow into the test site by adding a buffer containing the conjugate to the area of the conjugate source.

According to a preferred embodiment, the conjugate contains protein A conjugated to colloidal gold. In other embodiments, the conjugate comprises an antibody, preferably a monoclonal antibody conjugated to colloidal gold. Advantageously, colloidal gold has a particle size of from about 20 to about 80 nm.

According to another embodiment, the test strip further comprises a control portion located on the membrane downstream of the test portion. The control site preferably contains a control binder that is able to bind the conjugate. For example,

if the analyte of interest is an antibody and the conjugate contains an antibody-binding substance, such as a protein, then the control binder may be rabbit IgG (IgG = immunoglobulin G). If the analyte of interest is an antigen and the conjugate contains a monoclonal antibody that is specific for the antigen, then the control binder may be, for example, a protein.

The test strip may contain one or more additional components. The buffer pad may have fluid communication with the region of the conjugate source, so adding a wash buffer to the buffer pad causes the conjugate to flow out of the region of the conjugate source and flow through the membrane to contact the test site. The absorbent strip and absorbent pad can be located downstream of the membrane to absorb excess buffer as it passes through the membrane. A desiccant tablet may also be in close proximity to the membrane to absorb excess liquid and moisture.

The test strip, if necessary, can be placed in the housing, for example, in plastic. The housing preferably comprises a sample window located on top of the test and control sections, and a buffer window located upstream of the test section.

A method for detecting an analyte in a liquid sample, which involves applying a sample to a test portion of the membrane and then contacting the test portion with a conjugate that is capable of binding the analyte of interest and creating a visual signal indicating binding.

Brief Description of the Drawings

1 shows a test board.

Figure 2 shows a top view of the test board.

Figure 3 shows a side view of the board.

Figure 4 shows a section of the components that form the test strip in accordance with the first embodiment.

5 shows a test board with a positive result indicating that the analyte of interest was detected in the sample.

6 shows a test board with a negative result indicating that the analyte of interest was not detected in the sample.

7 shows a test board with a sample cup.

Detailed description of a preferred embodiment of a utility model. Diagnostic devices are available for quickly determining the presence of analyte in a sample. Although these devices are mainly described with reference to an immunodiagnostic device for detecting antibodies or antigens in a sample, those skilled in the art will readily understand that the device can easily be modified to detect any analyte of interest.

Previously, in immunodiagnostic devices, pre-mixing of the conjugate with the sample to be tested for the presence of antibodies was usually used. For example, in some devices, the dried conjugate was dissolved by adding a liquid sample, which allows pre-mixing of the sample and conjugate. The antibody to be detected, if any, is bound to the conjugate and can be visualized downstream of the test site containing the immobilized antigen. However, in this

In the system, non-specific interaction of the conjugate with other antibodies or other compounds in the sample leads to damping of the conjugate signal. As a result, a large amount of conjugate is required. Due to the use of a large amount of conjugate, a large reaction time is required before it is completed (purification), which increases the time required to obtain test results. In addition, the sensitivity of the test is so low that a significant amount of antigen is required at the test site to obtain a visual positive signal.

The device allows to solve the problems of the prior art by directly contacting the test site with the sample, which allows any analyte of interest to bind specifically with the immobilized binder. For example, an antibody of interest may be associated with an immobilized antigen. The conjugate is then fed to the test site along with the buffer. A buffer dissolution front pushes unbound compounds to the test site and reduces non-specific interaction with the conjugate. As a result, the required amount of conjugate is significantly reduced and due to this the reaction rate increases :. In addition, the sensitivity of the reaction increases, which allows the use of less antigen in the test area.

The devices and methods described herein can be used, for example, to diagnose diseases or disorders of a patient, such as human immunodeficiency virus (HIV) or hepatitis virus. They can also be used to determine a patient’s pregnancy. Other possible applications will become apparent to those skilled in the art from the description below.

Definitions

An “analyte of interest” is a molecule or compound whose presence must be identified in a sample. Analysts may be

without limitation, viral antigens, bacterial antigens, hormones such as insulin, follicle stimulating hormone (FSH), thyrotropin, relaxin, growth hormone and gonadotropin, enzymes, immunoglobulins, cytokines, drugs, cancer antigens, antigenic polysaccharides and nucleic acids. Specialists will easily understand that other analytes can be identified in samples using the proposed methods and devices. Particularly preferred analytes of interest are an anti-HIV antibody, an anti-HCV antibody (HCV = hepatitis C virus), and human chorionic gonadotropin (hCG).

The term “conjugate” refers to a composition that is configured to bind to an analyte of interest and generates a distinguishable signal. The conjugate usually contains a binder component conjugated to the label. The binder component allows the conjugate to bind to the analyte of interest and, possibly, to the control compound. The label creates a distinguishable signal, but mainly a visual signal. In accordance with one of the options, a visual signal is created only after binding of the conjugate to the analyte. In a preferred embodiment, the analyte of interest is an antibody, and the conjugate contains a binding component that is capable of binding antibodies. For example, the first binding component may be a protein, although other molecules that are capable of binding antibodies can be used. In another embodiment, the analyte of interest is an antigen, and the conjugate binding component comprises an antibody that is capable of binding to the antigen. Preferably, the antibody is a monoclonal antibody.

A label is any molecule or compound that can be attached to or conjugated to a binding component and which

can create a distinguishable signal. The preferred label is colloidal gold. Alternatively, the label may be, for example, dry latex particles, colloidal silver or other colloidal metals, colloidal carbon black, or other known compounds. Advantageously, the label has a particle size that does not impair the ability of the binder component to bind the analyte of interest. For example, when the binding component is protein, the label preferably has a particle size of approximately 5 to 120 nm, and more preferably approximately 20 to 80 nm.

The conjugate is advantageously prepared in a “conjugate buffer” which can stabilize and protect the conjugate. In one embodiment, after preparation of the conjugate in buffer, the conjugate can be dried on a “conjugate pad,” as discussed in more detail below. A conjugate that is dried on a support is considered "diffusely coupled." A conjugate is “diffusely coupled” if it is able to diffuse or flow, for example, by contacting it with a buffer. For example, a conjugate that has been diffusely coupled to the region of the conjugate source by drying can be dissolved in a buffer and can therefore flow along the membrane of the test strip.

A “control conjugate” is a conjugate in which the binder component is specific for the control compound. The control conjugate is advantageously used when it is likely that the entire conjugate will be bound to the analyte on the test line and not be sufficient for control. The control compound is usually the compound present in the sample to be analyzed, which is capable of binding the control binder to the control strip. Possibly, the binder component of the control conjugate may be specific for the compound that is present in the control region of the test membrane.

The conjugate is predominantly present in the "region of the conjugate source." The conjugate source region typically contains a conjugate that is diffusely coupled to it, such as a dried conjugate. Alternatively, the conjugate source region may comprise a conjugate that has been suspended in a buffer, such as a wash buffer or conjugate buffer. In one embodiment, the conjugate source region is a conjugate pad containing a dried conjugate that is fluidly coupled to a test membrane. In another embodiment, the conjugate source region is a portion of a test membrane on which the conjugate has been dried. In another embodiment, the conjugate source region is a buffer pad into which a buffer containing a conjugate can be introduced that is fluidly coupled to the test membrane. The conjugate source region is usually located upstream of the test site on the test membrane, so that after administration of the solution containing the conjugate or dissolution of the diffusely coupled conjugate, the conjugate flows through the test membrane and contacts the test site.

The conjugate is predominantly dissolved in a “wash buffer,” as discussed below in more detail. In short, the wash buffer is a buffered solution that predominantly contains a "blocking agent." Blocking agents are well known per se and contain any molecules or compounds that reduce non-specific interactions, such as non-specific antibody binding. Preferred blocking agents are proteins such as casein and bovine serum albumin (BSA). Other blocking agents that can be used are commercially available agents that are well known in the art.

A “sample buffer” is a buffered solution that

predominantly free of conjugate. In some embodiments, the sample buffer contains a blocking agent, while in other embodiments it does not. The sample buffer may optionally be applied to the membrane prior to injection of the sample, as discussed in more detail below.

A “test membrane” is a solid substrate having a test portion and possibly a control portion. The sample to be analyzed is usually applied directly to the test membrane so that it contacts the test site and the control site. The test membrane can be any solid support with which an analyte binding agent and a control binding agent can be coupled. Advantageously, the test membrane is made of nitrocellulose.

The test membrane provides a spreading drop of liquid in the radial direction. The test membrane and other components of the test strip are said to be in fluid communication if a fluid, 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 fluidly coupled. However, those skilled in the art will readily understand that direct contact between two specific components is not required for fluid communication.

The terms "test site," "test strip" and "test line" are used interchangeably and refer to the area on the test membrane to which the analyte binding material is linked.

An “analyte binding material,” “specific binding partner” or “binding component” is any molecule or compound that is configured to provide a specific bond to an analyte of interest. For example, and without any limitation, the analyte binding material may contain antigens, antibodies, receptors, other polypeptides,

peptides, haptens, lectins, nucleic acid containing oligonucleotides, or small molecules. In one embodiment, the analyte binding material is an antigen that is specific for the antibody to be detected in the sample. In another embodiment, the binding compound is an antibody that is specific for the antigen of interest.

The terms “control portion,” “control strip” and “control line” are used interchangeably and refer to the region on the test membrane to which the control binder is adhered.

A “control binder” is any molecule or compound that is capable of providing a specific bond to a conjugate or to a control conjugate.

A "test strip" is a complete device that can be used to detect the presence of analyte in a sample. The test strip advantageously comprises at least one test membrane with a test section and a control section. As discussed in more detail below, the test strip may be installed in a case, such as a plastic case. Alternatively, the test strip may be used without a housing. When installed in a chassis, the test strip and chassis together can be called a "test board."

A “test board window” or “sample window” is a hole in the plastic case of the test board through which a sample can be introduced. The test board window is predominantly located above the test section and the control section. The test board window also advantageously allows visualization of test results.

“Buffer window” refers to the hole in the plastic case of the board

testing through which a buffer can be entered. In accordance with one embodiment, the buffer window is advantageously positioned in such a way that it allows the buffer to be applied to the buffer pad. According to another embodiment, the buffer window is positioned in such a way that it is possible to apply the buffer directly to the test membrane.

"Volume indicator window" refers to the hole in the plastic case of the test board through which you can see a visual indicator of the sample volume. The volume indicator window is located downstream of the sample window. A sample volume indicator indicates that an acceptable sample volume is in contact with the test site. The sample volume indicator can be, for example, methylene blue or another colored component that is applied to the membrane directly upstream of the volume indicator window. The sample volume indicator moves through the volume indicator window due to sample movement, creating a visual signal in the window. In other embodiments, the sample volume indicator is a compound that changes color upon contact with the sample, such as a pH indicator. Typically, a sample volume indicator is used when a large sample volume is needed, for example, when it is believed that the analyte of interest is present at a low concentration in the sample. For example, an indicator of sample volume is predominantly used when urine is analyzed for the presence of hCG.

“Breakdown” refers to any material that is subject to analysis for the presence of an analyte of interest. The sample is predominantly in liquid form. Examples of samples include, but are not limited to, body fluids such as whole blood, plasma, serum, saliva, and urine. As discussed in more detail below, the sample may be solid. However, in this case, the sample is predominantly dissolved or extracted before its use.

A “sample application area” is a location on a membrane in

which put the sample. The sample application area is preferably located downstream of the conjugate source area and upstream of the control site. Preferably, the sample application area contains a test site. The sample can be applied directly to the sample application area, for example, using a pipette, or indirectly, for example, through a wick that is in contact with the membrane.

The term “antibody” is used broadly and includes, for example, and without limitation, whole antibodies, as well as a single antibody chain, antibody fragments, and chimeric antibodies, provided that they retain the desired binding specificity.

Testing Board

The diagnostic devices disclosed herein advantageously comprise a test board 1, as shown in FIGS. 1-3. Figure 1 shows the test board 1, which contains a plastic case 10 with an upper section 15 and a lower section 18, which are connected by snap-in to each other. There are two holes in the upper portion 15 of the plastic case 10: the sample window 20 and the buffer window 30. The sample window 20 allows the sample to be applied to the membrane sample application zone, which is located inside the plastic case 10. The sample application zone mainly contains a test section 140. Control section 150 predominantly located downstream of test portion 140 on the membrane. The buffer window 30 allows the buffer to be applied to the conjugate source region 50. The membrane is part of the test strip, which is described in more detail below.

In some embodiments, an optional optional volume indicator window (not shown) is used, which is located downstream of the control area. The volume indicator window, if any, allows the user

visualize an indicator, such as a color change, on the membrane downstream of the control site. The indicator indicates that a sufficient volume of sample has been brought into contact with the test site, so that the user can start contacting the test site with the conjugate to detect bound analyte, as discussed in more detail below, for example, by applying a buffer to the area of the conjugate source.

Figure 2 shows a top view of the test board 1, where you can see the window of the sample 20 and the window of the buffer 30 inside the plastic housing 10. Access to the test portion of the membrane 140 is possible through the window of the sample 20, and access to the source area of the conjugate 50 is possible through the window of the buffer 30 The control section 150 is located downstream of the test section 140. Although the window of the sample 40 is shown rectangular and the window of the buffer 30 is shown round, it should be borne in mind that these windows can be of any shape and size. However, their shape and size are advantageously chosen so as to direct the sample to the application area of the membrane 40 and direct the buffer to the source area of the conjugate 50. In addition, the sample window is configured to allow visualization of the test section 140 if a positive test result is obtained , and control plot 150.

Figure 3 shows a side view of an exemplary test board 1. The upper portion 15 and the lower portion 18 of the plastic housing are shown. The expanded end (60) of the upper portion 15 of the plastic housing, which is removed from the buffer window (not shown), is designed to accommodate absorbent material such as an absorbent pad or desiccant tablet on a test board (1). The size of the expanded end (60) may be different depending on the amount of absorbent material used, which, in turn, depends on the sample volume and buffer used in the test.

Test strip

The test strip contains a test membrane with at least one test site. An analyte binder (analyte binder) that is specific for the analyte of interest is bound or otherwise immobilized at the test site. At least one portion (zone) of the test portion becomes visible upon completion of the test if the analyte of interest is present in the sample. In a specific embodiment, the test site contains an immobilized antigen, and the test allows the identification of a specific antibody in the sample, such as biological fluid. In another embodiment, the test site contains an associated antibody and the test allows the identification of a specific antigen in the sample.

The sample is applied to the sample application area, which is located downstream of the area of the conjugate source, for example downstream of the conjugate pad. According to a preferred embodiment, the sample application area comprises a test site. However, in other embodiments, the sample application area is located upstream of the test site and the sample flows from the sample application area to the test site.

A preferred embodiment of the test strip is shown schematically in FIG. 4. Test strip 100 to check the sample for the presence of an analyte of interest, such as an antibody, preferably contains a buffer pad 110 that is in contact with the conjugate pad 120. In turn, the conjugate pad 120 is in contact with the test membrane 130, which contains the test portion 140 and the control portion 150. In turn, the membrane 130 is in contact with the absorbent pad 160. The absorbent pad 160 may be in contact with additional absorbent paper 170.

The absorbent pad 160 and / or the optional absorbent paper 170 may be in additional contact with the optional desiccant 180, for example, a desiccant tablet.

In some embodiments, a removable filter holder 190 is attached to the test strip 100. The filter holder 190 includes filter paper 200. When the filter holder 190 is attached to the test strip 100, the filter paper 200 is located directly above the sample application area, which advantageously contains the test section 140 and the control section 150.

A sufficient sample volume should be applied to the test site to ensure a positive visual reading after completion of the test, if the analyte is present in the sample. Thus, analytes that are suspected to be in the sample at a low concentration, for example, antigens such as hCG in the urine, require a larger sample volume. Test strip 100 may be configured to receive large volumes of sample. One such embodiment is shown in FIG. 7, where the sample cup 500 is attached to the test strip 100. The sample cup 500 comprises a container that contains a volume of liquid sample. Typically, the sample cup has an opening at the bottom through which the sample can flow out of the sample cup 500 and contract with the test membrane 130. Thus, the sample cup 500 is positioned so that the sample flows from the sample cup 500 into the region of the applied sample the membrane 130 and is in contact with the test sections 140 and the control section 150. Advantageously, the sample application area comprises a test section 140 and a control section 150, and the sample cup 500 is located directly above the test section m 140 and a control section 150. The sample cup 500 may optionally have a filter through which the sample passes earlier

contacting the test membrane.

The sample can be introduced into the sample cup after attaching it to the test board.

In another embodiment, the sample is first introduced into the sample cup, and then the cup is attached to the test board and conditions are created for the sample to flow into the sample application area.

It is desirable to ensure that the sample volume containing the detectable amount of analyte, if any, is brought into contact with the test section 140 prior to allowing the conjugate to flow into the test section 140. A sample volume indicator, such as an indicator dye, located downstream of the test section 140 to the control section 150, can be used to determine the fact that a sufficient amount of sample was brought into contact with the test section 140 and the control section 150. For example, a dye dye, such as methylene blue, can be applied to absorbent pad 160 or to absorbent paper 170 downstream of the test membrane. The flow of a liquid sample through an absorbent pad 160 or absorbent paper 170 causes a dye to flow and creates a visual signal, for example, a visible blue color. This signal can be observed through the volume indicator window on the test board (not shown).

In some embodiments, especially those in which a large sample volume is to be applied (approximately over 50 μl), the conjugate pad 120 is not in fluid communication with the test membrane 130. One such option is shown in FIG. 7. Separation of the conjugate pad 120 and the test membrane 130 prevents the conjugate and the sample from mixing before contacting the sample with the test portion 140. After contacting the sample with the test portion 140 and the control portion 150, the conjugate pad 120 is moved and introduced into

fluid communication with the test membrane 130. The buffer is then applied to the buffer pad 110 to start the test. The buffer can be applied to the buffer pad 110 directly through the buffer window, or by cutting the buffer ampoule 300, whereby the buffer can flow into the buffer pad 110. In one embodiment, the buffer is applied to the buffer pad 110 before moving the pillow 120 conjugate and its introduction into fluid communication with the membrane 130. In accordance with alternative options, the buffer is applied directly to the pillow 120 of the conjugate.

The method by which the conjugate pad 120 is forced to move and enter into fluid communication with the membrane 130 is not limited. In one embodiment, the user shifts the movable portion of the housing to which the conjugate pad 120 and, optionally, the buffer pad 110 are attached, whereby the conjugate pad 120 contacts the membrane 130. The mechanism for moving the conjugate pad 120 to create fluid communication with The test membrane 110 advantageously also causes the buffer ampoule 300 to burst. This mechanism may also remove the sample cup 500 from the test strip 140.

The desiccant tablet 180 is preferably in fluid communication with the absorbent paper 170. The desiccant tablet size 180 is chosen so as to absorb the full volume of the sample deposited on the test membrane 130. The desiccant tablet and any other absorbent materials are located downstream of the sample application area and are exposed sample flow through the test site.

In other embodiments, the sample cup is replaced with a wick that is in contact with the liquid sample. The sample flows along the wick to the sample application area of the test membrane and is in contact with the test section and the control

plot. In a specific embodiment, the wick is placed in a stream of urine to analyze urine for the presence of an analyte such as hCG. In these embodiments, the conjugate pad is not predominantly in fluid communication with the test membrane during application of the sample to the wick. After a sufficient volume of sample has come into contact with the test site, the conjugate pad is moved to make contact with the test membrane. The conjugate pad movement mechanism may also serve to remove the wick. The buffer is then introduced into the buffer pad or directly into the conjugate pad to begin the test. The buffer may be inserted directly into the buffer pad through the buffer window, as already described above. Alternatively, the buffer may be in the ampoule on the test board and conditions may be created for it to flow to the buffer pad and / or to the conjugate pad by cutting the ampoule or otherwise opening it.

The test strip is preferably located inside the housing, as shown in FIG. 2 and described hereinabove.

Figure 5 shows a positive test result, indicating the presence of the analyte of interest in the sample. The test portion 140 of the membrane 130 is visible as a line through the sample window 30. In addition, the control section 150 is visible as a line through the sample window 20, which indicates that the conjugate flowed from the conjugate source region 50 through the test section 140. For comparison, Fig. 6 shows a negative result. With a negative result, only the control section 150 is visible as a line through the window 20 of the sample. And in this case, the control indicates that the conjugate was flowing from the region 50 of the conjugate source through the test section 140. Thus, the absence of a line on the test section 140 is the result of the absence of a bound analyte of interest.

Using a test strip

The sample, which is checked for the presence of a specific analyte of interest, is applied to the region of the applied sample of the test membrane so that it contacts the test site. According to a preferred embodiment, the sample is applied to the membrane and conditions are created for contact with the test site before contacting the conjugate with the test site. Thus, the analyte of interest in the sample, if any, has the ability to bind to the analyte binding substance in the test site, previously binding to the conjugate.

If the test strip is in the housing, then the sample is preferably applied through the sample window. In this case, the membrane contains the area of application of the sample, access to which is provided through the window of the sample. Advantageously, the sample application area contains at least one positive test site. Preferably, the application area comprises a test section and a control section. Alternatively, the sample may be applied through a wick or cup, so that it can flow into the application area of the membrane sample and come into contact with the test and control sites.

If the test strip is not in the housing, the sample can be applied directly to the application area of the membrane sample, mainly containing the test section and the control section. Alternatively, the sample may be applied to the membrane at the application area upstream of the test site and may flow through the membrane to contact the test site. Advantageously, however, the sample is contacted with the test site before the conjugate is introduced into the membrane.

According to the first embodiment, the sample is accumulated in a sample cup or

injected into it. The cup is attached to the casing and conditions are created for the sample to flow to the area of application of the membrane sample and for contact with the test section and the control section. The sample cup is advantageously used when the sample volume is approximately greater than 10 μl. The sample cup may optionally contain a filter through which the sample is passed before contacting with the membrane.

In accordance with an additional option, the sample is applied to the wick, which is in contact with the membrane. The wick allows the sample to migrate to the area of application of the membrane sample and to contact the test and control sites. The wick mainly contains fiberglass paper, polyester, cellulose or other material that allows the sample to migrate to the membrane containing the test site. If the test board is in the case, the wick can protrude from the case, which provides access to the wick, for applying the sample. For example, if you want to determine the presence of a specific analyte in the urine, such as hCG, then the wick can be introduced into the urine stream of the patient being tested. In another embodiment, the wick may be brought into contact with a sample to be analyzed, for example, with bodily fluids such as saliva.

The test sample is preferably applied to the application area of the membrane sample in liquid form. Exemplary test samples may be, for example, (but not limited to), serum, plasma, whole blood, saliva, and urine. Solid or semi-solid samples can be dissolved and applied to the membrane, or extracted prior to application.

In accordance with other options, the sample buffer, which mainly does not contain conjugate, is applied to the membrane before applying the sample. In some

variants of the sample buffer contains a blocking agent. Advantageously, the sample buffer is applied to the membrane at the site of application of the sample. Approximately 1 to 25 μl of sample buffer is preferably applied to the membrane, preferably, approximately 1 to 10 μl, and even better, approximately 1 to 5 μl. In a specific embodiment, 5 μl of the sample buffer is applied to the membrane sample application area, after which 5 μl of serum, plasma, whole blood or other liquid sample is applied. Adding sample buffer before applying the wash buffer is particularly preferred when testing for hepatitis C virus, as discussed in more detail below, and when viscous samples are used. Using a sample buffer increases the sensitivity of the test and causes viscous samples to flow faster.

The sample buffer mainly contains a buffering agent and an anticoagulant. Optionally, it may contain one or more detergents and / or sugars. Any buffering agent can be used, preferably in an amount that maintains the desired pH of the sample buffer. Advantageously, the pH of the sample buffer is approximately 7.2 to 7.6. For example, and without limitation, a buffer can be selected from the group consisting of HEPES, Tris, and phosphate buffers. According to a preferred embodiment, the sample buffer contains approximately 0.01% to 0.5% HEPES, and even better, approximately 0.02 to 0.05% HEPES. In accordance with another embodiment, the sample buffer contains approximately 0.01% to 0.5% phosphate, and even better, approximately 0.02 to 0.05% phosphate. Those skilled in the art will readily understand how to adjust the amount of the buffering agent to maintain the desired pH.

The sample buffer also preferably contains an anticoagulant such as sodium azide or Proclin. According to a preferred embodiment, the sample buffer contains approximately 0.1% to 1.0% sodium azide, more preferably

approximately from 0.1% to 0.2%, and even better, mainly about 0.2% sodium azide.

Depending on the nature of the sample to be tested, the sample can also be optionally applied to the membrane through a filter. Exemplary sample materials that are predominantly filtered include whole blood, fecal samples, urine, food extracts, and waste extracts. In one embodiment, whole blood is applied through a membrane filter. The filter removes contaminants and allows analytes, such as antibodies, to pass through it, thereby increasing the sensitivity of the test. The filter is preferably inserted into a holder, which is attached to the test board or to the test strip in the required manner. When attaching to the test board, the filter is preferably attached directly above the sample window. The filtering agent is selected based on the type of sample to be filtered. Specialists will easily understand how to make such a choice. For example, for whole blood samples, the filter material may be (but not limited to) Pall BTS-SP300 ^TM .

As mentioned above, the filter is advantageously attached to the test strip using a circuit filter holder, however, for specific applications, the filter can be permanently attached. The filter holder is usually a device that snaps onto the body of the test board. The design and type of device may be different, but usually it is made of polystyrene, polyethylene, polypropylene or other plastic.

A test portion is a portion of a membrane that is coated with a binder, preferably a protein, that is capable of specifically binding to an analyte of interest. For example, a positive test site may be coated with antigen, which is

specific for the antibody of interest. In accordance with specific options, the test site contains HIV-associated antigen or hepatitis antigen, as described below in more detail in the Examples. When the test site contains antigen, approximately 0.025 μg to 0.4 μg of antigen is preferably used.

In other embodiments, an antibody that is specific for the antigen of interest is immobilized in a test site. The antibody may be a polyclonal or monoclonal antibody. When the test site contains an antibody, approximately 0.1 to 20 μg of antibody is preferentially applied, preferably approximately 0.2 to 2 μg, and even better, approximately 0.5 to 1 μg. In a specific embodiment, the test site contains an antibody for hCG, and preferably a monoclonal antibody for hCG.

A binder may be applied to the membrane in accordance with any desired pattern; thus, the visible result of a positive test can take any form. According to a preferred embodiment, the test section is linear and forms a line extending from one side of the membrane to the other, so that a vertical line appears that runs across the window of the sample if a positive result is obtained, indicating the presence of analyte in the sample. In other embodiments, the test site is in the form of a symbol, for example, such as "+."

You can test several types of analyte in a single test strip. In this case, prepare additional positive test sites on the test membrane. For example, the second positive test site may contain an antigen for a second type of antibody, or an antibody for a second type of antigen. Binder on

the additional positive test site is not necessarily a binder of the same type as in the first test site. So, for example, the binder in the first test site can be an antibody, while the binder in the second test site can be an antigen. In this way, a sample can be analyzed for the presence of several types of analyte.

Each additional positive test site may have a shape different from that of the first test site to allow the user to easily discern the nature of the positive result. Depending on the type of additional analytes to be tested, additional conjugates that bind different analytes may be needed. For example, if the analytes of interest are two different antibodies, then a single conjugate containing a protein can be used. However, if one analyte is an antibody and the second analyte is an antigen, then two different conjugates will be required, one of which specifically binds the antibody and the other specifically binds the antigen.

The control site preferably contains a control binder that will bind to the conjugate. Since the control site is located downstream of the positive test site, the reaction in the control site will indicate that the conjugate migrated through the test site, where it was for the reaction in the presence of the analyte of interest. If the conjugate is intended for antibody recognition, the control region preferably contains immunoglobulin, such as rabbit IgG. If the conjugate contains an antibody, then the control region preferably contains an antibody-binding substance, such as a protein. The control portion may have any desired shape, such as a line or symbol, for example, “-.”

In other embodiments, the control site contains a binder that binds to the control conjugate. For example, a control site may contain β-gal, and a control conjugate may contain β-galactose conjugated to gold.

The binder and the control binder can be applied to the test membrane using any known method. They are usually applied by contacting the test membrane with a binder, for example, by spraying or contacting the test membrane using a pump distribution system. Such systems are commercially available, for example, supplied by Kinematic Automation.

When the sample comes into contact with the test site, and if the analyte of interest is present in the sample, it will bind to the binder in the test site. Excess unbound analyte is washed off the test site by migration of the buffer along the membrane, as discussed in more detail below.

After applying the sample to the membrane, a wash buffer is applied to the test membrane. Advantageously, approximately 1 to 20 is applied first, and even better, approximately 1 to 5 drops of the wash buffer are approximately. An additional wash buffer may be applied later if the reaction needs to be cleaned.

The wash buffer primarily carries (conveys) the conjugate to the test site. The wash buffer can be applied directly to the test membrane. However, in a preferred embodiment, the wash buffer is applied upstream of the buffer pad from where it flows into the test membrane. The buffer pad may be made of any material that may contain a buffer and allows it to flow through the pad into the membrane at the desired speed. For example, a buffer pad may contain

one or more materials selected from the group consisting of fiberglass paper, cellulose and polyester. According to a preferred embodiment, the buffer pad is Whatman GF / DVA fiberglass paper. Those skilled in the art will easily understand how to choose the appropriate material for the buffer cushion, taking into account factors such as the design of the test strip casing with the spreading of a liquid droplet in the radial direction, the desired flow rate and the amount of buffer to be held in the buffer cushion.

The wash buffer may be applied directly to the buffer pad, for example, using a pipette or dropper. However, in another embodiment, an appropriate amount of the wash buffer is present in a container located in close proximity to the buffer pad. For example, the wash buffer may be stored in a glass or plastic ampoule. After the introduction of the test sample, the container containing the washing buffer is cut or otherwise opened, which allows the washing buffer to contact the buffer pad.

The composition of the wash buffer may vary to increase the sensitivity of the test. Advantageously, the wash buffer contains a buffering agent, an anticoagulant, a detergent, sugar and other materials that can reduce background and / or non-specific binding.

Any buffering agent that maintains the desired pH of the buffer, preferably comprising approximately 7.2 to 7.6, can be used. For example, and without limitation, the buffer may be selected from the group consisting of HEPES, Tris, and phosphate buffer. According to a preferred embodiment, the washing buffer contains approximately 0.01% to 0.5% HEPES, more preferably approximately 0.02 to 0.2% HEPES, and even better,

approximately from 0.02 to 0.05% HEPES. In a particularly preferred embodiment, the wash buffer contains 0.025% HEPES.

The wash buffer may also contain an anticoagulant such as sodium azide or Proclin. According to a preferred embodiment, the wash buffer contains approximately 0.1 to 0.5% sodium azide, and preferably approximately 0.1 to 0.2% sodium azide.

Sodium chloride or a similar role is predominantly present at a concentration of about 1%, and even better, about 0.85%. EDTA can be predominantly present at a concentration of about 0.1%. Sugar such as mannitol may also be present. Preferred mannitol concentrations are from about 1% to 5%. In a preferred embodiment, the concentration of mannitol is about 1%.

A blocking agent is also predominantly present in the wash buffer. The blocking agent may be any known blocking agent. An advantageous blocking agent is a protein such as casein or bovine serum albumin (BSA). The blocking agent is predominantly present in the wash buffer at a concentration of approximately 0.01 to 0.5%, and even better, approximately 0.02 to 0.2%. In a particularly preferred embodiment, casein is present in the wash buffer at a concentration of about 0.1%.

The wash buffer typically also contains a detergent, preferably a non-ionic detergent, such as Triton X100 ^™ or Tween. Mostly, the detergent is present at a concentration of approximately 0.05 to 1%, and even better, approximately 0.1 to 0.5%. In a particularly preferred embodiment, the wash buffer contains Triton X100 at a concentration of about 0.1%.

The pH of the wash buffer is adjusted so that it is

approximately from 7 to 8, and mainly approximately from 7.2 to 7.6. In a particularly preferred embodiment, the pH of the wash buffer is approximately 7.2.

The test strip also contains the region of the conjugate source. According to a preferred embodiment, the test strip comprises a conjugate pad on which the conjugate has been dried, as already described above. The wash buffer flows from the buffer pad to the conjugate pad with which it is in contact. The conjugate pillow, in turn, is in contact with the test membrane. However, in some embodiments, the conjugate pad is contacted with the membrane after adding a sample. In particular, when a large sample volume is used, the conjugate pad is preferably brought into contact with the membrane after the sample is contacted with the test site.

A sufficient volume of the wash buffer is applied to the buffer pad to dissolve the conjugate diffusely coupled 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 can be determined by those skilled in the art based on 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. Advantageously, 1 to 10 drops of the buffer solution are applied to the buffer pad when the buffer pad is approximately 2 cm × 5 mm in size, and preferably 2 to 5 drops, and even better, 3 to 4 drops of the buffer solution.

In alternative embodiments, a conjugate pad is not used. In accordance with one alternative, the conjugate is present in the wash buffer at a concentration in the range of approximately 0.2 OD / ml to 1.0 OD / ppm. The conjugate-containing wash buffer may be applied to the buffer pad. Thus, the conjugate flows with the wash buffer from the buffer pad.

to the membrane where it contacts the test site and the control site. In another embodiment, a conjugate-containing wash buffer is applied directly to the test membrane. The wash buffer containing the conjugate may be stored in the ampoule, and may leak to the membrane after cutting or otherwise opening the ampoule. The wash buffer containing the conjugate is preferably applied to the membrane at a point upstream of the control site. However, in some embodiments, a conjugate-containing wash buffer is applied directly to the test site after application of the sample.

In another embodiment, the conjugate is diffusely coupled, for example, by drying, directly to a membrane that contains a test section and a control section. The wash buffer can then be applied to a buffer pad that is in contact with the membrane, or it can be applied directly to the test membrane. The wash buffer dissolves the dried conjugate and carries it along the test membrane to the test site and the control site.

The wash buffer containing the conjugate predominantly contacts the test site after the sample has already been applied, and any analyte present in the sample already has the ability to bind the immobilized binder. Thus, the wash buffer is preferably applied to the test strip after applying the sample to the test site.

The conjugate described in detail hereinabove is prepared for use in the test by dilution in conjugate buffer. Advantageously, a working conjugate is prepared by diluting the conjugate in the conjugate buffer to approximately 3 OD / ml. The conjugate buffer composition may vary in

depending on the nature of the conjugate. An exemplary conjugate buffer is one such as HEPES, Tris, phosphate, or other similar buffers known per se. A preferred conjugate buffer contains approximately 0.02 to 0.2% HEPES buffer, and preferably about 0.025% HEPES. The conjugate buffer may also contain a salt, preferably sodium chloride. Sodium chloride is added at a concentration of approximately 0.1%, and preferably about 0.85%. The conjugate buffer may also contain an anticoagulant such as sodium azide or Proclin at a concentration of about 0.1 to 0.2%.

The conjugate buffer also predominantly contains EDTA (about 0.1%), casein at a concentration of about 0.02 to 0.2%, and mostly about 0.15%, cow's serum albumin (BSA) at a concentration of about 0.5 to 3%, and mostly about 1%, mannitol at a concentration of approximately 1% to 5%, and preferably about 1%, and sucrose at a concentration of approximately 1% to 5%, and preferably about 1%. Triton X100 can also be present in the buffer, mainly at a concentration of approximately 0.1 to 0.5%, and even better, about 0.1%.

The conjugate buffer is preferably adjusted so that its pH is approximately from 7.2 to 7.6, and preferably about 7.2.

In one embodiment, after preparing the working conjugate by diluting the conjugate in the conjugate buffer, the working conjugate is dried on a conjugate pad, for example, by drying under vacuum. The conjugate pad is preferably cut into strips of the appropriate size for use as test strips. The following examples use strips of 5 mm × 105 mm in size, obtained by cutting the conjugate pad.

The conjugate pillow mainly contains fiberglass, paper from

fiberglass, polyester or cellulose. Such materials are known per se and can be purchased, for example, from Millipore, S&S and Whatman. In a preferred embodiment, the conjugate pillow is a commercially available fiberglass conjugate pillow type GFCP203000 (Millipore).

As already described above, the wash buffer dissolves the dried conjugate and causes it to flow out of the conjugate pad and along the test membrane through the test section and the control section.

The test membrane may be made of any material with which a protein or other binding agent may adhere, covalently or non-covalently, and through which a conjugate with a wash buffer may flow. The test membrane is mainly made of nitrocellulose. Specialists may use other membranes with higher or lower flow rates as test membranes based on factors such as desired sensitivity, test time, and cost.

When the conjugate flows with the wash buffer through the membrane, it contacts the test site. If the analyte of interest is present and bound to the binder in the test site, the conjugate in the buffer will be bound to the analyte.

A conjugate-containing wash buffer that was not bound at the test site will continue to flow through the membrane to the control site. The conjugate will bind to a control binder. The wash buffer will also carry (flush) an unbound sample from the test and control sites.

A wash buffer containing an unbound conjugate and an unbound sample will then flow into the absorbent pad that is in contact with

the downstream end of the membrane. The absorbent pad may be made of any known absorbent material. Advantageously, the absorbent pad is absorbent paper, such as S&S 900 ^TM paper. Other absorbent papers are known that can be selected by specialists depending on the final configuration of the test strip.

The wash buffer may continue to flow through the absorbent pad and flow into the absorbent paper that is in contact with the absorbent pad. Absorbent paper is mainly used when it is necessary to absorb a large amount of buffer.

A desiccant tablet forming a molecular sieve can also be integrated into the test strip. In one embodiment, a buffer with an unbound conjugate flows from absorbent paper into a desiccant tablet with which the paper is in contact. A desiccant tablet traps the absorbed liquid and eliminates the reverse flow into the test strip.

A desiccant tablet may also keep the test strip dry until used. A preferred desiccant tablet is 0.395 g. Tri-Sorb, which can be purchased from Sud-Chemie (product number 43-01).

Specialists will easily understand how to choose the appropriate size of the absorbent pad and the material for its manufacture, absorbent paper and desiccant tablet, depending on the final configuration of the test strip, including the full amount of sample and buffer to be used.

The test strip as a whole is predominantly located inside the case, such as a plastic case. As already described here above, in one embodiment, the housing contains a sample window through which the sample can be introduced into the membrane containing the test and control sections. Housing can also

contain a buffer window through which the buffer can be applied to the buffer pad or to the membrane. The housing may also contain a volume indicator window through which you can see a signal indicating that a sufficient volume of sample is contained in the test participation. A sufficient sample volume is the sample volume, which is considered to contain a sufficient amount of analyte of interest, if any, that creates a visual signal at the end of the test if the analyte is present in the sample. For example, in the urinary hCG detection test, approximately 200 µl of urine is sufficient sample volume.

The test results are mainly taken into account after approximately 1 to 5 minutes after adding the wash buffer to the test strip. In accordance with one embodiment, the results are read after approximately 2 to 4 minutes after adding the wash buffer, and preferably approximately after 3 to 4 minutes after adding the wash buffer. In a particularly preferred embodiment, the results are read after 1 minute after the addition of the wash buffer. As shown in FIG. 7, in the case of a positive result, the test line (300) becomes visible. A visible control strip (400) indicates that the test was performed correctly. As shown in FIG. 6, if the control line (400) is visible and the test line is not visible, this indicates a negative result.

Example 1 Construction of an HIV Test Board

A quick visual test was prepared with a fluid droplet spreading in a radial direction to detect antibodies to the human immunodeficiency virus. The construction of the test strips mainly corresponds to that shown in FIG.

Test strips have a plastic base (6 cm × 5 mm) with glue on

both sides. The plastic base is approximately 2 to 20 mils thick.

A test line and a control line were formed on a nitrocellulose membrane (Millipore HF09004) by spraying using a pump distribution system (Kinematic Automation). The test line is a vertical line of HIV antigen. HIV antigen is 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 glycoprotein antigen (GP36 (about 0.5 mg / ml), at a concentration of approximately 0.5 to 1.0 mg / ml prepared in PBS (styrene-butadiene copolymer) containing 5% trehalose A total amount of approximately 0.1 to 0.5 μg of antigen was applied to the test site in a vertical line.

Other test strips were also prepared that contain only HIV-1 or HIV-2 antigen on the test line.

For all test strips, the control section contains a vertical line of the control antibody. The control antibody is rabbit IgG diluted to 1 mg / ml in PBS containing 5% trehalose. A sufficient amount of the control antibody was applied to the control site, sufficient to create a visible control line after conjugate binding.

For each test failure, the corresponding nitrocellulose membrane was cut into strips of approximately 2.5 cm × 5 mm in size, which were attached to a plastic base,

Protein A bound to colloidal gold, which can be purchased from Sigma, was used as a conjugate for testing. The conjugate was diluted in conjugate buffer to a concentration of approximately 3 OD / ml. Conjugate buffer contains 0.25% HEPES, 0.85% sodium chloride, 0.1% azide

sodium, 0.1% EDTA, 0.1% casein, 1% albumin, cow's serum, 1% mannitol, 5% sucrose, and 0.1% Triton X100. The buffer was adjusted to pH 7.2.

The conjugate was applied to a fiberglass conjugate pad (Millipore GFCP203000), which was dried and cut into pieces of 5 mm × 5 mm. These pieces were attached respectively to the end of the cellulose membrane closest to the test site of each test strip.

Whatman GF / DVA fiberglass paper buffer pad (2 cm × 5 mm) was then attached to the dried conjugate pad of each test strip.

An absorbent pad (2 cm × 5 mm) made of S&S 900 paper was attached to the end of the nitrocellulose membrane closest to the control area, with an additional piece of writing paper (2.5 cm × 5 mm; S&S 470) attached to the absorbent pad. In turn, the absorbent paper was in contact with a 0.395 g desiccant tablet (Tri-Sorb, Sud-Chemie).

The test strip was placed in a plastic case to get a test board similar to that shown in FIGS. 1-6. The plastic case (10) has a test window (20) that opens the test section (140) and the control section (150), as well as a buffer window (30) located directly above the buffer cushion. The plastic case has labels to indicate the location of the test line and control line. Test boards were individually packaged in foil.

Example 2 HIV Testing

Serum or plasma was prepared from a whole blood sample taken from a patient tested for HIV using the appropriate venipuncture technique. Samples were stored at temperatures from 2 ° C to 8 ° C and used for 24 hours or frozen at -20 ° C for use for 2 weeks.

Frozen samples were thawed before use.

The test board described in Example 1, which contains a mixture of HIV-1 and HIV-2 antigens at a control site, was removed from the packaging in the HIV foil.

About 5 μl of sample buffer containing 0.025% HEPES and 0.2% sodium azide were applied to the membrane through the center of the window of the test board.

The sample pipette, which contains the serum or plasma to be tested, was held upright over the test window of the test board and 5 μl of the sample was introduced into the membrane in the center of the window.

Four drops of the washing buffer solution (0.025% HEPES, 0.85% sodium chloride, 0.1% EDTA, 1% mannitol, 0.1% casein, 1% Triton X100, pH 7.2) were introduced into the buffer pad. After 3 minutes, if the reaction was not completed, another drop of buffer was added to the buffer pad.

The test results were read after a period of 1 to 5 minutes after the introduction of the first buffer. As shown in FIG. 5, a positive result is two visible lines running through the sample window: a test line (140) and a control line (150). A positive result is obtained if both lines are visible, even if one of them is darker than the other. The negative result is only the control line visible through the sample window, as shown in Fig.6.

Example 3 Test Board for Hepatitis C Virus

A test strip for the diagnosis of hepatitis C virus (HCV) was prepared as described in Example 1. However, a mixture of hepatitis C antigen, which contains the core (0.3 mg / ml), NS3 (0.4 mg / ml) and NS4 (0.1 mg / ml) sprayed in the form of a test line on the membrane to obtain a test site, instead of HIV antigen. A total of about 0.4 μg of the hepatitis C antigen mixture was applied as a test line. Rabbit IgG diluted to 1 mg / ml

sprayed as a control line.

Example 4 Test for hepatitis C virus

The test board described in Example 3, which contains the HCV antigen at the test site, was removed from the HCV foil package.

About 5 μl of sample buffer, which contains phosphate buffer and 0.2% sodium azide, was applied to the membrane through the center of the window of the test board.

Serum or plasma was prepared from a whole blood sample. A sample pipette that contains the serum or plasma to be tested was held upright above the test window and the test board was injected into the membrane in the center of the window 5 μl.

Four drops of the washing buffer solution (0.025% phosphate, 0.85% sodium chloride, 0.1% EDTA, 1% mannitol, 0.1% casein, 1% Triton X100, pH 7.2) were introduced into the buffer pad. After 3 minutes, if the reaction was not completed (not purified), another 2 drops of buffer were added to the buffer pad.

The test results were read after a period of 1 to 5 minutes after the introduction of the first buffer.

As shown in FIG. 5, a positive result is two visible lines running through the sample window: a test line (140) and a control line (150). A positive result is obtained if both lines are visible, even if one of them is darker than the other. The negative result is only the control line visible through the sample window, as shown in Fig.6.

Example 5 Pregnancy Test

Pregnancy tests are quick visual tests with a fluid drop spreading radially. The construction of the test strips mainly corresponds to that shown in Fig.7.

Each test strip contains a plastic base (6 cm × 5 mm) with

glue on both sides. The plastic base is approximately 2 to 20 mils thick.

A test line and a control line were formed on a nitrocellulose membrane (Millipore HF09004) by spraying using a pump distribution system (Kinematic Automation). The test line is a vertical line of an antibody to human chorionic gonadotropin (hCG), a predominantly monoclonal antibody. Antibodies to hCG are widely known and can be purchased, for example, from Research Diagnostics Inc. (RDI-CBL74; New Jersey, USA) and Charles River Labs, or can be prepared using standard procedures. The total amount of approximately 0.2 to 1 μg of the antibody is applied to the test site in the form of a vertical line.

The control line contains a vertical line of protein A. The total amount of protein A sufficient to create a visual signal after conjugate binding, which is applied to the control line, is usually about 0.25 μg.

For each test strip, the corresponding nitrocellulose membrane was cut into strips of approximately 2.5 cm × 5 mm in size, which were attached to a plastic base.

A second anti-hCG antibody was coupled to colloidal gold to give a conjugate for testing. Gold-hCG colloidal conjugates can be purchased from Research Diagnostics Inc. Alternatively, hCG antibody conjugates may be prepared using known techniques (see, for example, US Pat. No. 6,485,982, which is incorporated herein by reference). The conjugate is diluted in conjugate buffer to a concentration of about 3 OD / ml. The conjugate buffer predominantly contains 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 pH 7.2.

The conjugate was applied to a fiberglass conjugate pad (Millipore GFCP203000), which was dried and cut into pieces of 5 mm × 5 mm.

Whatman GF / DVA fiberglass paper buffer pad (2 cm × 5 mm) was then attached to the dried conjugate pad of each test strip.

An absorbent pad (2 cm × 5 mm) made of S&S 900 paper was attached to the end of the nitrocellulose membrane closest to the control area, with an additional piece of inlet paper (2.5 cm × 5 mm; S&S 470) attached to the absorbent pad. In turn, the absorbent paper was in contact with a desiccant tablet (Tri-Sorb, Sud-Cheinie), which is capable of absorbing a substantial portion of the sample volume and, thus, drawing the sample and analyte through the test section.

Methylene blue, or another indicator, is applied to an absorbent pad or absorbent paper downstream of the test site. The movement of this volume indicator indicates that a sufficient sample volume has come into contact with the test site, which allows for accurate test results.

The test strip is placed in a plastic case to get a test board. The plastic case has a test window above the test section and a control section, which provides visualization of the test results. The case also has a volume indicator window through which you can observe the movement of the volume indicator.

On some test boards, the sample cup is mounted above the test window. The sample cup has a sample container with a graduation

a hole allowing the sample to flow into the sample application area of the test membrane through the sample window. The sample cup also has an outlet filter. After introducing the sample into the membrane, the cup is removed so that the test results can be seen.

Other test boards use a wick that protrudes from one end or one side of the chassis. The wick creates a path along which a sample can flow into the application area to the test site.

The housing also has a mechanism that allows the conjugate pad to be moved to create fluid communication with the membrane.

In some test boards, an ampoule containing a wash buffer is also housed in a housing upstream of the conjugate pad. In other test boards, the buffer is introduced into the buffer pad through the buffer window.

Starting the conjugate pad movement mechanism to create contact with the test membrane cuts the ampoule, allowing the buffer to flow into the conjugate pad. Advantageously, about 5 drops of the wash buffer are contacted with the conjugate pad. The start of the movement mechanism also removes the sample cup or wick from the body.

The plastic case has labels to indicate the location of the test line and the control line in the sample window.

Example 6 Pregnancy Test with Sample Cup.

Quick visual tests for the presence of pregnancy with the spreading of a drop of liquid in the radial direction, passable with a cup for the sample, is carried out in accordance with the procedure described in Example 5.

Approximately 500 μl of patient urine is injected into the sample cup, which is attached to the test board above the sample window. When a blue color appears in the volume indicator window, the mechanism starts, which creates a fluid connection of the pillow

conjugate with a membrane. In this case, cut the ampoule containing the buffer, and begin the test. The sample cup is removed and visual test results are observed in the sample window after 1-5 minutes.

As shown in FIG. 5, a positive result is two visible lines through the sample window: a test line 140 and a control line 150. The result is considered positive if two lines are visible, even if one of them is darker than the other. The negative result is one control line 150, which is visible through the sample window, as shown in Fig.6.

Example 6 Pregnancy Test with Wick

Quick visual tests for the presence of pregnancy with the spreading of a drop of liquid in the radial direction, carried out with a wick, carried out in accordance with the procedure described in Example 5.

The patient inserts the end of the wick into her stream of urine. The blue color that appears in the volume indicator window indicates that a sufficient sample volume has been brought into contact with the test site.

The start of the mechanism in the housing introduces the conjugate pad into fluid communication with the membrane. Cut the ampoule containing the buffer and start the test when the buffer flows to the conjugate pad. The wick is removed and visual test results are observed in the sample window after 1-5 minutes.

As shown in FIG. 5, a positive result is two visible lines through the sample window: a test line 140 and a control line 150. The result is considered positive if two lines are visible, even if one of them is darker than the other. The negative result is one control line 150, which is visible through the sample window, as shown in Fig.6.

Although some preferred embodiments of the utility model have been described, it is clear that those skilled in the art

areas may be amended and supplemented, which do not however go beyond the framework of the utility model formula. In addition, numerous modifications can be made in order to adapt the proposed solutions to a specific situation, without going beyond the scope of the utility model.

Claims (36)

1. A diagnostic test strip for detecting an analyte of interest in a liquid sample, comprising a conjugate source region that contains a conjugate configured to bind an analyte of interest; a sample application area located downstream of the indicated area of the conjugate source and configured to receive a liquid sample; and a test site located downstream of the conjugate source region and containing an immobilized binder specific for the analyte of interest. 2. The test strip according to claim 1, in which the area of application of the sample contains a test section. 3. The test strip according to claim 1, in which the analyte of interest is an antibody. 4. The test strip according to claim 3, in which the analyte of interest is an antibody for human immunodeficiency virus (HIV). 5. The test strip according to claim 3, in which the analyte of interest is an antibody for hepatitis C virus (HCV). 6. The test strip according to claim 1, in which the analyte of interest is an antigen. 7. The test strip of claim 6, wherein the analyte of interest is human chorionic gonadotropin (hCG). 8. The test strip according to claim 1, in which the region of the source of the conjugate contains a dried conjugate. 9. The test strip of claim 8, in which the region of the conjugate source comprises a conjugate pad that contains dried conjugate and is in fluid communication with the test strip. 10. The test strip of claim 8, in which the source region of the conjugate is a portion of the test strip. 11. The test strip according to claim 1, in which the cause of the flow of the conjugate to the test site is the contacting of the source region of the conjugate with the wash buffer. 12. The test strip of claim 11, wherein the wash buffer contains a protein blocking agent and a non-ionic detergent. 13. The test strip of claim 12, wherein the wash buffer contains 0.1% casein and 1% Triton XI 00. 14. The test strip according to claim 1, in which the conjugate contains a binder component and a label. 15. The test strip of claim 14, wherein the binder component is a protein. 16. The test strip of claim 14, wherein the label is selected from the group consisting of colloidal gold, colloidal silver, colloidal soot and dried latex particles. 17. The test strip of claim 16, wherein the label is colloidal gold. 18. The test strip according to 17, in which the colloidal gold has a particle size of from 20 to 80 nm. 19. The test strip according to claim 3, in which the conjugate contains a protein conjugated to colloidal gold. 20. The test strip according to claim 6, in which the conjugate contains an antibody conjugated to colloidal gold. 21. The test strip according to claim 1, in which the test strip contains a membrane of nitrocellulose. 22. The test strip according to claim 1, which further comprises a control site located downstream of the test site, and the control site contains an immobilized control binder that is capable of binding the conjugate. 23. The test strip according to item 22, in which the control section contains immobilized rabbit IgG. 24. The test strip according to item 22, in which the control section contains immobilized protein. 25. The test strip according to claim 1, which further comprises a buffer pad that has fluid communication with the region of the conjugate source. 26. The test strip according to claim 1, which further comprises an absorbent pad, which is in fluid communication with the membrane in a position downstream of the test site. 27. The test strip according to claim 1, which further comprises a sample filter located directly above the area of application of the sample. 28. A test strip for detecting anti-HIV antibodies in a sample that contains a test section and a conjugate source region located upstream of the test section; an immobilized antigen bound at a test site, wherein the immobilized antigen contains from 0.1 to 0.5 μg of HIV antigen; and a conjugate diffusely coupled to said region of the conjugate source, wherein the conjugate is configured to bind an antibody. 29. The test strip of claim 28, wherein the HIV antigen contains one or more antigens selected from the group GP120, P24, and GP36. 30. The test strip according to p, in which the conjugate contains protein A. 31. The test strip of claim 30, wherein Protein A is conjugated to colloidal gold. 32. An immunodiagnostic pregnancy test strip containing a sample application area for receiving a liquid sample; a test site containing an immobilized first anti-hCG antibody; and a conjugate source region comprising a conjugate configured to bind hCG, wherein the conjugate source region is located upstream of the sample application region and from the test site. 33. The pregnancy test strip of claim 32, further comprising a wick that is fluidly associated with the sample application area. 34. The pregnancy test strip of claim 32, wherein the conjugate contains a second hCG antibody. 35. The pregnancy test strip of claim 34, wherein the second hCG antibody is conjugated to colloidal gold. 36. A test device for conducting a pregnancy test, comprising the immunodiagnostic test strip of Claim 32, and a urine collection cup.