JP2000055918A - Antibody assay device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device for measuring or detecting an antibody in humor, especially a device that accurately, simply, and specifically detects an antibody being directed toward the source of infection existing in urine as a clinical specimen. SOLUTION: An antibody assay device is provided with at least a test site 5 where ligand for capturing a target antibody being assayed is immobilized (i), and a control site 6 where the ligand for capturing an arbitrary antibody in a sample to be inspected is immobilized (ii) in a second region 3 (b) in an assay device including a solid-phase support where a first region 1 (a) for allowing the sample to be inspected to contact and the second region 3 (b) for allowing an antibody in the sample to be inspected to react are provided so that the sample to be inspected is transported from the first region 1 to the second region 3 by a capillary phenomenon, and/or a labeling method that detects a reaction result in the second region 3.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for detecting or measuring an antibody present in a body fluid, and more particularly to an apparatus for detecting an antibody directed to a pathogen such as a bacterium or a virus present in urine as a clinical specimen. The present invention relates to a device that can be easily and specifically detected or measured. Further, the present invention relates to a solid-phase assay method capable of accurately detecting and measuring a target antibody present in a body fluid, particularly, urine.

[0002] According to the present invention, false negatives caused by, for example, a thin test sample can be distinguished from true negatives, so that a highly accurate antibody assay can be performed with less erroneous analysis. Therefore, it is particularly useful for an assay targeting a specimen having a small absolute amount of an antibody such as urine.

[0003]

2. Description of the Related Art Detection and measurement of specific antibodies directed to pathogens such as bacteria and viruses present in body fluids are useful for indirectly diagnosing the presence or absence of infection. An immunoassay method and a measurement apparatus using a pathogen or a component derived therefrom as an antigen (ligand) of a measurement system are used.

[0004] One of the methods is to perform a binding assay (antigen-antibody reaction) on a single piece of porous substrate in the form of a strip. This type of assay utilizes the property that the liquid applied to one end of the strip sequentially moves in a capillary in a certain direction due to the capillary characteristics of the porous substrate. That is, when a test sample (liquid) containing an analyte is attached to one end of a strip in which various reaction components are sequentially arranged at specific positions, the test sample sequentially moves along a capillary along the strip, Thereby, the reaction components come into contact with each other at a desired position and react. The presence of the analyte is also confirmed and measured by detecting a signal from a detectable label involved in the binding reaction between the ligand and the receptor.

[0005] Immunoassays using these principles are:
It is often called an immunocapillary assay or an immunochromatographic assay, and is disclosed in, for example, WO 87/02774, EP 0306772, and the like. An improved method is disclosed in JP-A-64-638.
65, JP-A-1-294964 and JP-A-6-167497.

[0006] Such a conventional assay method has an advantage that the measurement can be completed in a short time without the need for a measuring instrument or the like, but there is still room for improvement in terms of sensitivity and specificity. In addition, since each test is an independent measurement, negative or positive control samples cannot be measured at the same time, and it cannot be determined whether the obtained measurement values are reliable based on appropriate measurements. Have a problem.

[0007] In particular, when the test sample is a body fluid other than blood such as urine, the amount of antibodies contained therein is very low, usually about 1/1000 to 1 / 10,000 of the amount of antibodies in blood. Concentration. In addition, there is considerable variation in the amount of antibody between samples, such as urine samples from subjects who have consumed large amounts of water, such as extremely thin.

[0008] In this case, in the above-mentioned conventional assay system,
"Negative" when the test sample is too thin to detect the antibody
In order to show the results, there was a problem that whether the results were "negative" being true or "negative (false negative)" because the test sample was too thin could not be distinguished. In addition, when a test sample with a small amount of antibody such as urine is targeted, a measurement system that can detect with high sensitivity is required, but as a result, non-specific reactions are easily detected and false positives are likely to be shown. There are also problems.

[0009] Urine as a biological sample, compared to blood,
Due to its non-invasive, convenient and safe preparation, it is considered to be a particularly desirable specimen for clinical examination. Therefore, an antibody assay system that provides sufficient accuracy even when urine is used as a sample, that is, an assay system with high accuracy and high specificity, in which false negatives and false positives are reduced, is required.

[0010]

SUMMARY OF THE INVENTION The present invention is an improved method of the above immunocapillary assay or immunochromatographic assay developed in view of the above circumstances. The present invention relates to a method for detecting the presence of a target antibody to be analyzed in a test sample. An object of the present invention is to provide an analysis technique capable of accurately detecting and measuring the amount by distinguishing between “false negative” and “true negative” caused by a cause based on a test sample. It is another object of the present invention to provide an analysis technique for suppressing non-specific reactions derived from a test sample and reducing false positive detection in an immunocapillary assay or an immunochromatographic assay (← Added .).

Thus, the present invention provides an excellent antibody measurement technique (an apparatus useful for performing an assay, which provides sufficient accuracy even when a body fluid having a relatively small amount of antibodies such as urine is used as a test sample. It is intended to provide an assay using the device.

[0012]

Means for Solving the Problems The present inventors have made intensive studies to solve the problems in the above antibody measurement technique and to develop a highly accurate apparatus for measuring urine antibodies using urine as a test sample. As a result, in addition to the site for detecting the target antibody to be analyzed (the "test site"), any antibody contained in the test sample is detected and measured in the reaction region (determination region) of the strip. It has been found that by providing a "control site", "true negative" can be accurately detected and measured separately from "false negative". In other words, according to such an assay system, when an unsuitable test sample that cannot be determined because the concentration of the test sample is low or the like is used, the amount of total antibody in the test sample is small, so that the control site is Indicates negative (indeterminable display). On the other hand, if the test sample is appropriate, the control site shows positive, and the result at the test site in this case indicates the measurement target (target antibody) in the test sample.
, That is, whether it is “positive” or “true negative”.

It should be noted that Japanese Patent Application Laid-Open Nos. 1-294964 and 6-16749 which disclose improved methods of immunocapillary assay (immunochromatographic assay).
No. 7 discloses an assay device in which a control site is provided in addition to a test site in a determination area as in the present invention. To determine whether or not it has moved and passed the test site, which is completely different from the present invention.

In addition, when the present invention is applied to a specimen having a low sample concentration such as urine and a large amount of contaminants, the present inventors conducted a binding reaction between an antibody to be measured and a ligand thereto in the presence of a component derived from Escherichia coli. It has been found that the non-specific reaction originating from the sample can be suppressed by performing the above. Furthermore, by using a reagent having binding specificity to the Fc region of IgG as an antibody detection reagent in a method for detecting a urinary antibody by an antigen-antibody reaction, nonspecific reactions derived from the sample can be suppressed and false positive detection can be reduced. Was found.

The present invention has been completed based on such findings; at least (a) the first method of bringing a test sample into contact with a test sample;
A solid support having a region and (b) a second region for reacting an antibody in the test sample so that the test sample is transported from the first region to the second region by capillary action; and An assay device containing a labeling means for detecting a reaction result in two regions, wherein (b) a test site in which (i) a ligand for a target antibody to be assayed is immobilized, ii) An antibody assay device having a control site on which a ligand for capturing any antibody in a test sample is immobilized.

In particular, a feature of the antibody assay device of the present invention resides in that a control site is provided separately from the test site in the second region, and the control site is provided with an appropriate device for applying an appropriate test sample. If properly measured in, regardless of whether the target antibody is present in the test sample,
Form a positive result indicator in the presence of the label, while the presence of the target antibody in the test sample occurs when an improper test sample is applied or measured with an improper instrument or improperly. Then, regardless of whether or not the label is configured to form a display indicating a negative result in the presence of the label.

That is, the control site in the second region of the device of the present invention has a test site result (particularly a negative result) regardless of whether the target antibody is present in the test sample.
This corresponds to a portion for displaying whether or not the assay result is valid. Thus, based on such a configuration, according to the antibody assay device of the present invention, it is possible to distinguish between false negatives and true negatives and accurately and qualitatively and quantitatively determine the antibodies in the test sample.

Further, the antibody assay device of the present invention is characterized in that the reaction of the antibody at the test site is designed to be carried out in the presence of a component derived from Escherichia coli, and / or
Alternatively, it is designed such that a reagent having binding specificity to the Fc region of the target antibody IgG is used as desired as an antibody detection reagent for detecting a reaction at the test site. Based on such a configuration, according to the antibody assay device of the present invention, it is possible to suppress nonspecific reactions and qualitatively and quantitatively determine the antibody in the test sample with high sensitivity and high accuracy.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The assay device of the present invention is improved so as to accurately detect and measure the presence and / or amount of a target antibody to be analyzed in a body fluid using a solid-phase assay. is there.

In the present invention, the “body fluid” is not particularly limited as long as it is a body fluid of a living body (including humans and other animals) in which the presence of the antibody to be measured is recognized. These include, but are not limited to, bodily fluids such as urine, spinal fluid, amniotic fluid, saliva, blood including serum and plasma, and preferably bodily fluids other than blood. In particular, the present invention solves the problem of detection accuracy in urine specimens that have been conventionally desired to be used for antibody detection.In view of the effects of the present invention, urine is particularly preferably exemplified as a body fluid. it can.

The "antibody" to be measured present in the body fluid is not particularly limited as long as it is an antibody whose measurement or detection is desired, and is, for example, an antibody directed to various pathogens which are foreign substances to the living body. be able to.

The pathogen is not particularly limited.
Examples include various pathogens that infect humans and other animals and consequently produce antibody. Specifically, for example, HIV (human immunodeficiency virus), A
Hepatitis virus such as hepatitis virus, rubella virus, influenza virus, measles virus, cytomegalovirus, herpes simplex virus, varicella / zoster virus, adenovirus, enterovirus, etc .; pylori), bacteria such as Chlamydia, Mycobacterium tuberculosis, Spirocheta, Neisseria gonorrhoeae, Syphilis, Mycoplasma, etc .; Preferably, viruses such as HIV, various hepatitis viruses, rubella virus, influenza virus, measles virus, herpes virus; H. pylori and the like, and particularly preferably H. pylori. Bacteria such as H. pylori.

According to the present invention, there is first provided a solid support having at least a first region and a second region.

The first region is a region to which a test sample is attached and which comes into contact with the test sample, and the second region is a region where an antibody (a whole antibody which may include a target antibody) contained in the test sample is a ligand-receptor or an antigen. -A region (reaction region and judgment region) that reacts and binds in the relation of an antibody and further shows the reaction result in the presence of a label. These regions are applied to a first region of the solid support, and are arranged on the solid support in such a manner that the contacted test sample is transported by capillary action from the first region to the second region. . Preferably, all or at least a part of the test sample in contact with the first region is arranged to be capillary-migrated to the second region through the substantially planar layer of the solid support.

The solid support may have a third region following the second region as a region for absorbing a test sample (liquid) that moves by a capillary from the first region through the second region. good.

The solid support preferably has the shape of a strip, and the first area and the second area are such that the test sample is placed on the same plane of the strip by capillary action from the first zone (first area) to the second area. It is arranged in such a way that it moves to a band (second region) and, if necessary, further flows into a third band (third region) following the second band. The preferred shape of the solid support is a strip shape as described above, but any other shape may be used as long as the shape and the form can perform various functions of the solid support of the present invention. Can be adopted.

The solid support can absorb a test sample (liquid), and when wetted by the sample, at least the antibody contained in the sample is removed from the first region of the solid support by the capillary action. It can be made to flow into two regions and, if necessary, further from the second region to the following third region. Further, the solid support is preferably capable of supporting and immobilizing a ligand which reacts with and captures an antibody (including a target antibody) contained in the test sample.

Examples of suitable solid supports include porous substrates such as polyethylene, glass fiber, cellulose, rayon, nylon, cross-linked dextran, various types of chromatography paper, nitrocellulose, filter paper and the like.

Each of the first, second, and third regions of the solid support can be composed of the same or different base materials selected from the base materials listed above. Various selections are adopted according to the role and function of the.

The first region of the solid support is preferably made of a porous substrate capable of absorbing a test sample applied to the surface thereof and allowing the sample to be capillary-migrated to the second region. The porous substrate suitable for the first region is not particularly limited, but is usually polyethylene (for example, POLEX: Po
rex Tchnoogies, Fairburn, Georgia), cellulosic materials including glass fiber, rayon, nylon, and paper. Preferably porous polyethylene,
It is a cellulosic material such as filter paper.

The second region of the solid support can be moved by the capillary while wicking the test sample (liquid) from the first region to the second region by its capillary action. It is desirable that the porous substrate be capable of supporting a ligand for an antibody (including a target antibody) so as not to be detached by capillary movement of a sample. Such porous substrates include filter paper, chromatography paper, glass fiber, cross-linked dextran, nylon, nitrocellulose, and the like, preferably, for the reason that the ligand can be easily immobilized.
Nitrocellulose.

The second region has a test site formed by binding and immobilizing a ligand for specifically recognizing and capturing a target antibody in a test sample, and recognizing and capturing any antibody in the test sample. A control site formed by binding and immobilizing a ligand is provided. The control site is arranged at a certain distance from the test site, preferably at a position downstream of the capillary movement, and it is desirable that both sites are brought into contact with the liquid tip of the test sample under the same conditions.

The ligand at the test site is not particularly limited as long as it specifically binds to the target antibody to be analyzed. Preferably, the ligand is specifically recognized by the target antibody to be analyzed and specifically An antigen that binds (antigen-antibody reaction) can be mentioned. As such an antigen, an antigen used in an existing serum antibody measurement system can be widely used. These may be the above-mentioned pathogens such as viruses and bacteria, but may be at least those having an antigenic determinant unique to the pathogen. For example, pathogens inactivated by heating or irradiation, antigens obtained by extracting the pathogens with surfactants, antigens artificially prepared by chemical synthesis or genetic engineering techniques, etc. Can be exemplified.

The ligand at the control site is not particularly limited as long as it binds to any antibody contained in the test sample. Preferably, the ligand specifically recognizes any antibody contained in urine. Antibodies that bind to antibodies (anti-
Immunoglobulin antibody).

These ligands are immobilized on the porous substrate located at the test site and the control site, respectively, so as not to be detached and removed from the test site and the control site due to the capillary movement of the liquid sample. That is, each ligand binds to each site of the porous substrate so as not to diffuse when the second region is wetted by the sample containing the analyte, and remains immobilized at each site, It is desirable that the solid support is supported so as not to be transported to the third region.

The immobilization of such a ligand can be achieved by a physical bond or a chemical bond to the above-mentioned various porous substrates by a method known in the art.

For example, diazo method, peptide method (acid amide derivative method, carboxyl chloride resin method, carbodiimide resin method, maleic anhydride derivative method, isocyanate derivative method, cyanogen bromide activated polysaccharide method, cellulose A carbonate derivative method, a method using a condensation reagent, etc.), an alkylation method, a carrier binding method using a crosslinking reagent (for example, using glutaraldehyde, hexamethylene isocyanate, etc. as a crosslinking reagent), a carrier binding method using a Ugi reaction, etc. Chemical reaction: or ionic bonding method: physical adsorption method and the like. When the porous substrate used in the second region is nitrocellulose, the ligand can be easily bound by non-covalent bonding.

The amount of the ligand (antigen) used for the test site in the second region is preferably an excess amount so that essentially all of the target antibody which is considered to be present in the test sample binds to the test site. .

The amount of the ligand (anti-immunoglobulin antibody) used for the control region of the second region is as follows:
It is preferred that any antibodies in the test sample (which may include the target antibody) are in excess so that essentially all bind to the control site.

It is preferable that the binding reaction between the target antibody and the ligand (particularly the antigen) at the test site is performed in the presence of a component derived from Escherichia coli. Such a component derived from Escherichia coli may be used in a reaction system by being mixed in a diluted solution of a test sample and being applied to the first region together with the test sample, or may be used in a test site of a solid support or an upstream region thereof. (The first area described above, a tracer area described later, etc.) may be attached and fixed so as to be detachable. The component derived from Escherichia coli may be any of those described below, but preferably includes a soluble extract of Escherichia coli and lipopolysaccharide (LPS).

When the test sample is applied to the first region, the sample flows from the first region into the second region by capillary movement, where the target antibody in the sample is first bound and immobilized on the test site in the second region. Then, any remaining antibodies (including target antibodies that could not bind to the test site) are bound and immobilized on the control site located downstream thereof.

The labeling means used in the present invention is used as a means for detecting whether or not the target antibody and any antibody in the test sample are bound and immobilized on the test site and the control site, respectively.

Examples of such a labeling means include those comprising a ligand for an antibody and a detectable label bound to the ligand.

The ligand for the antibody is not particularly limited as long as it is a molecule that recognizes and binds to the antibody present in the test sample. In the present invention, the ligand in the target antibody to be measured and the test sample are not limited. It is desirable that the antibody binds to any of the contained antibodies, and has the same binding property as the ligand of the control site described above, particularly, the same binding property as that employed as the ligand of the control site. An example is an anti-immunoglobulin antibody.

The anti-immunoglobulin antibody includes, for example, an antiserum obtained from any immunized animal immunized with an immunoglobulin corresponding to the target such as human immunoglobulin or a purified product thereof (polyclonal antibody) or a monoclonal antibody. Antibodies are included.

It should be noted that the anti-immunoglobulin antibody as a ligand for the control site is, if desired, directed to all classes of antibodies so as to capture and detect the total antibody contained in the test sample. It is possible,
Alternatively, it can be directed to any desired class of antibodies, such as immunoglobulin G (IgG). Preferably, such a ligand can be an anti-immunoglobulin antibody directed against an antibody of the same class as the class of the target antibody desired to be measured, in which case detection of the target antibody and alloantibody is performed at the control site. This is preferable in that not only urine but also various body fluids such as serum are used for a test sample in that the most suitable index is provided for judging whether or not the assay has been appropriately performed.

In the measurement of a urinary antibody in which the antibody to be measured is IgG, the ligand as the labeling means is more preferably a ligand characterized by having binding specificity to the Fc region of IgG. For example, an Fc-specific anti-IgG antibody that does not react with the light chain or F (ab) region of IgG, or protein A or protein G that specifically reacts with the Fc region of IgG.
Can be preferably exemplified. These anti-immunoglobulin antibodies or ligands can be prepared according to a conventional method, and can also be obtained as commercial products.

The detectable label component is not particularly limited as long as it is known in the art to be used in a specific binding assay, particularly an immunoassay, or any detectable label that can be used in the future (“Monoclonal antibody”). Tatsuo Iwasaki et al., Kodansha Scientific, 198
4; Enzyme immunoassay, 2nd edition, Eiji Ishikawa et al., Medical Shoin, 1982, etc.).

Preferred markers are those that preferably change color at the test site or control site in the second region. Although not limited, it is more preferable that the color change can be visually recognized without requiring any measuring instrument. For example, various chromogens, for example, a fluorescent substance, an absorptive dye and the like can be mentioned, and more preferably, a particulate label containing a visually detectable marker can be mentioned.

Suitable particulate labels include particles of polymers (eg, latex or polystyrene), sacs, liposomes, metal gels (eg, colloidal silver or gold) or polystyrene dyes. Preferably, a metal gel such as a silver colloid or a gold colloid is used.

Such a label is prepared as a labeled ligand by chemically or physically binding to the ligand according to a conventional method, and these can also be obtained as commercial products.

The labeling means used in the present invention can be applied to the second region (including the test site and the control site) of the solid support to detect the result of the reaction with the antibody in the sample generated in the second region. Anything that can be labeled as
The existence mode is not particularly limited as long as the purpose is satisfied. For example, when the assay device of the present invention is a flow-through type device, the labeling means can be included in the reagent kit of the present invention as being separate from the solid support.

Preferably, the labeling means is supported and fixed on the solid support in a detachable manner, and more preferably, is supported and fixed on the solid support in a detachable manner upstream of the second region of the solid support. It is desirable that it is More preferred embodiments are
The solid support is supported by a region (hereinafter, referred to as a tracer region) located between the first region and the second region. In such a case, the test sample applied to the first area is wicked to the tracer area by capillary movement,
There, it is contacted with the labeled ligand to form the target antibody / labeled ligand-complex and the optional antibody / labeled ligand-complex, respectively. After passing through the tracer region, the test sample containing such a complex further moves to the second region by capillary movement. The ligand (antigen) located at the test site in the second region is specific for the target antibody, and the ligand (anti-immunoglobulin antibody) located at the control site is specific for any antibody. For this reason, the target antibody / labeled ligand-complex in the sample that has migrated by capillary is first captured at the test site, and then the remaining optional antibody / labeled ligand-complex (target antibody / labeled ligand) in the sample. -Including the complex) is captured at the control site.

The tracer region of the solid support allows the test sample to be transported from the first region to the second region together with the target antibody and any antibody by capillary movement, and the labeled ligand described above is transferred along with the capillary movement. The material is not particularly limited as long as it is made of a substrate that can be supported and fixed so that it can be detached. Usually, a porous substrate is used, and examples thereof include polyethylene, glass fiber, rayon, nylon, and cellulosic materials including paper and the like.
Preferably, it is a material that does not easily exhibit non-specific adsorption,
For example, glass fiber etc. which may be optionally treated with polyvinyl alcohol (PVA) can be mentioned.

A preferred embodiment of the present invention includes a solid support in which the tracer region and the test site in the second region are arranged at regular intervals.
As described above, by providing a certain region between the tracer region and the test site, the test sample in contact with the labeled ligand in the tracer region can reach the test site in the second region before reaching the test site. Mixes the antibody and the labeled ligand to promote the binding reaction between the antibody and the labeled ligand. That is, such an area is an incubation area (time, space) for binding of the target antibody and the optional antibody to the labeled ligand before the target antibody and the optional antibody in the sample come into contact with the test site and the control site, respectively. Is provided.

Further, the solid support of the present invention may have a third region following the second region.

After the test sample comes in contact with the second region through the first region and, if necessary, the tracer region, it moves through the second region by capillary movement and continues to move to the third region. That is, the third region functions as a region for receiving the liquid flowing from the second region, and the liquid is transmitted to the second region by capillary movement.

The third region may be any one that normally functions to receive the liquid-containing substance that has not bound at least in the second region, and furthermore, the capillary flow of the liquid sample (that is, the tip of the liquid) It may also have a site that goes to a predetermined completion zone on the solid support to signal the completion of the assay.

For this purpose, the solid support may be provided with a visible indicator zone containing a water-soluble dye such as erythrosin B, safranin O or phenol red in the downstream region of the second region. In this case, the liquid tip of the test sample crosses the second region and flows through the indicator zone as it enters the third region, where the dye located there is moved down by capillary movement of the test sample (liquid). The test sample is transported to the basin where the test sample passes through the first region, the tracer region, and the second region (test site, control site), and it can be detected and confirmed that the assay is completed.

The third region is not particularly limited as long as it is made of a substrate capable of absorbing a test sample (liquid), and examples thereof include a cellulosic material including polyethylene, rayon, nylon, and paper. Can be. Preferably, it is a cellulosic material containing paper or the like.

Hereinafter, the present invention will be described with reference to the accompanying drawings showing preferred embodiments of the antibody assay device and its components. Note that the device illustrated in the drawings is merely one embodiment of the present invention, and the present invention is not limited to these embodiments.

FIG. 1 is a schematic diagram of a strip-shaped solid support (60 × 5 mm) included in the apparatus of the present invention. FIG.
In the above, symbol 1 is a first region (16 × 5 mm, thickness 0.92 mm) to which a test sample is applied and which comes into contact with the test sample, and symbol 2 is a labeled ligand (eg, gold colloid-labeled anti-human Tracer region (5 × 5) where IgG antibody) is supported
mm, thickness 0.79 mm), and symbol 3 is a second region (18 mm × 5 mm) that reacts with the antibody in the test sample and displays the result.
mm, thickness 0.1 mm).
This is a third region (22 × 5 mm, thickness 1.46 mm) for absorbing the test sample that has moved through the region.

The thickness of the first area is usually 0.2 to 2 mm, preferably 0.8 to 1.2 mm, and the thickness of the tracer area is usually 0.2 to 1.5 mm, preferably 0.5 to 1.5 mm. 1 mm, the thickness of the second region is usually 0.03 to 0.2 mm, preferably 0.08 to 0.12 mm, and the thickness of the third region is usually 0.5 to 3 mm, preferably 1 to 3 mm. Although 2 mm can be mentioned, it is not limited to these.

In the second region (3), a test site (test line, about 1 × 5 mm) (symbol 5), in which a ligand that specifically binds to the target antibody is supported and immobilized, binds to an arbitrary antibody. A control site (control line, about 1 × 5 mm) (symbol 6) in which a ligand (anti-human immunoglobulin antibody) is supported and immobilized is arranged. The test part (5) is arranged at a fixed distance (about 6 mm) from the tracer area (2), and the control part (6) is arranged at a fixed distance (about 6 mm) from the test part (5). You. The test site reacts specifically with the target antibody in the test sample and functions to indicate the presence or absence of the target antibody in the presence of the label, while the control site is used to determine whether the test sample used is appropriate. It functions to indicate the presence or absence in the presence of the sign.

The first component constituting the solid support of the strip,
The members (substrates) of the region, the tracer region, the second region, and the third region may be joined in the longitudinal direction of the strip on which the sample moves, and the joining mode is not limited. The distal region in the longitudinal direction of the first region and the proximal region of the tracer region, the distal region of the tracer region and the proximal region of the second region, and the distal region of the second region and the proximal region of the third region are laminated. It is desirable to be joined in a state. More preferably, as shown in FIG. 1, the longitudinal distal region of the first region is laminated on the proximal region of the tracer region, and the longitudinal distal region of the tracer region is laminated to the proximal region of the second region. And the base region of the third region may be further laminated on the distal region of the second region. By having such a bonding mode, the sample attached to the first region can move smoothly in the longitudinal direction of the strip. Although the longitudinal width of the laminated area is not particularly limited,
It is desirable to be as small as possible, preferably 0.5 to 2
mm, more preferably about 0.8 to 1.2 mm. Note that the vertical position of each region of the laminated portion is not limited to this, and may be reversed.

Next, the principle of the assay using the apparatus of the present invention will be described with reference to FIG.

In the assay, a test sample suspected of containing the target antibody is first applied to a first region (1) of a solid support.

The test sample may be a body fluid such as urine as it is, or may be diluted with an appropriate diluting solution. The diluting solution is not particularly limited.
A buffer (for example, a citrate buffer, a phosphate buffer, a Tris buffer, an acetate buffer, a borate buffer, etc.) having a buffering capacity in the range of about 5 to 9, preferably about pH 6.5 to 8.5;
Surfactants and the like can be included.

Since the antigen-antibody reaction is carried out in the presence of a component derived from Escherichia coli, nonspecific detection is suppressed and false positive detection is reduced. It is desirable to use
Here, the component derived from Escherichia coli is Escherichia coli.
It is not particularly limited as long as it is a component derived from, and may be, for example, a protein component, a saccharide component, a lipid component, or a mixed component thereof. Preferably, a soluble extract of E. coli,
Alternatively, lipopolysaccharide (LPS) can be exemplified. The method for preparing the E. coli-derived component is not particularly limited, and various methods can be used. Usually, any Escherichia coli is grown and propagated in a medium suitable for the growth of the Escherichia coli, and the obtained cells are collected and subjected to physical means using an ultrasonic crusher or the like, or a surfactant or the like. It can be prepared as a soluble component (soluble extract) by crushing or solubilization. LPS can also be prepared by extraction with an organic solvent (for example, using a single solvent such as phenol, chloroform and ether, or a mixture of two or three solvents), and can also be prepared artificially by genetic engineering techniques. it can. In addition, commercially available products can be used simply.

The amount of the component derived from Escherichia coli to be mixed in the diluted solution is not particularly limited, but is 0.1 to 1 per μg of the ligand (antigen) in the reaction system, ie, the test region.
It is preferably prepared so as to have a ratio of about 00 μg, preferably about 0.5 to 50 μg.

The amount of the component derived from Escherichia coli to be mixed with the test sample is usually 5 μg / ml or more, preferably 5 to 5 μg / ml.
0 μg / ml, more preferably in the range of 10 to 30 μg / m. The addition of 50 μg / ml or more is not particularly limited, but the effect of the present invention can be achieved with the addition of 50 μg / ml or less.

The first area is wetted by applying the test sample to the first area (1). The test sample applied is
The tracer region (2) is moved into the tracer region (2) by capillary movement through the first region (1), where the labeled ligand (colloidal gold-labeled anti-human IgG antibody) supported detachably in the tracer region. Contact and react.

When an appropriate test sample is used,
When the target antibody is contained in the sample, both the target antibody and any of the antibodies contained in the sample bind to the labeled ligand in the tracer region (2), and the target antibody / labeled ligand-conjugate, respectively. Body and any antibody / labeled ligand-complex. After the test sample passes through the tracer region (2), each formed complex or uncomplexed labeled ligand is transported from the tracer region (2) to the downstream region together with the test sample. In a preferred embodiment, the labeled ligand that has not yet formed a complex is a test sample containing an antibody between the tracer region (2) and the test site (5) in the second region (3). While moving the capillary together, sufficient time (space) is provided for complex formation. Next, when reaching the test site (5) in the second region, the target antibody / labeled ligand-complex contained in the test sample at the site (5) binds to the ligand supported by the site and is immobilized. Be transformed into Next, the test sample is further moved to the downstream region by capillary movement, and any antibody /
The labeled ligand-conjugate binds to the ligand (anti-immunoglobulin antibody) at the site and is immobilized. Next, the second
The assay result can be indicated as positive by detecting the complex immobilized at the test site (5) and the control site (6) of the region based on the labeling component of the labeled ligand. On the other hand, when a sample containing no target antibody is used as a test sample, a target antibody / labeled ligand-complex that binds to the test site (5) is not formed. Is not detected (negative).

By the way, the negative result displayed at the test site (5) includes a negative (false negative) result due to a low concentration of the sample (that is, a small amount of total antibody in the sample) and a target in the sample. Both negative (true negative) results in the absence of antibody are included. Such a negative is the test site (5)
However, in the case of false negative, no control antibody (labeled ligand-complex) binding to the control site (6) is formed, so that the control region shows negative and true negative. In this case, since any antibody / labeled ligand-complex is formed, the control region shows positive, and the control site (6)
The difference between the negative and the positive of the test site can discriminate whether the negative result of the test site (5) is false negative or true negative. Furthermore, when the measurement is not properly performed with an appropriate device, such as when an inactivated labeled ligand is employed, the control site (6) shows a negative result similarly, thereby causing false Negative detection can also be prevented.

The liquid sample containing any unbound antibody, labeled ligand and the like is further moved to the second region (3) by capillary movement.
And continues to move to the third area (4) located in the downstream area.
If desired, an indicator zone can be provided, where the liquid front carries dye from the indicator zone to the completion zone, indicating that the liquid dye has passed through the third region and the assay has been completed.

The above-mentioned solid support is preferably packaged in the form of an assay device so as to be easily used.

An example of the assay device used in the horizontal direction is shown in FIG. Solid support (A) including first region (1), tracer region (2), second region (3) (including test site (5) and control site (6)), and third region (4) Is in a housing made of a suitable material (B)
To be placed. As such a housing material, a moldable plastic such as polystyrene is preferable,
It is also possible to use other materials, for example glass, metal or paper. The housing consists of an upper section (7) with several openings and a lower section (8), the solid support being arranged on the lower section of the housing and covered by the upper section from above.
Openings (9) and (1) in the upper section of the housing
0) are aligned along the arrangement of each region of the solid support,
Each corresponds to the first region (1) and the second region (3) of the solid support.

The opening (9) makes it possible to apply the sample to the first area (1) of the support (sample addition window). The opening (9) preferably has a raised side surface around the opening, such a side surface forming a well to promote the liquid sample to be dropped and permeated into the first region of the support. I do. The method for bringing the test sample into contact with the first region of the solid support is not particularly limited, but is dropped straight down from the sample addition window (9) of the apparatus so as to be perpendicular to the plane of the solid support. Is preferred.

The opening (10) is arranged so that the test site and the control site in the second region of the solid support can be visually observed, whereby the labeled ligand /
The presence or absence of the binding of the target antibody-complex and the presence or absence of the binding of the labeled ligand / any antibody-complex to the control site can be visually detected (detection window, judgment window). The opening (10) does not necessarily have to be open so as to include both the test site and the control site, and the test site and the control site of the solid support are separated by two separate detection openings. A window may be formed.

According to the assay device of the present invention, usually 45
℃ or less, preferably 4 to 40 ℃, more preferably 25 to
After applying the test sample under the temperature condition of about 40 ° C., several minutes to 3 minutes
By leaving the sample for 0 minute, preferably 5 to 20 minutes, the presence / absence and / or amount of the target antibody in the test sample can be accurately assayed.

When performing an antibody assay using the above-described antibody assay device of the present invention, it is convenient to use a reagent kit including the device.

The present invention also provides a reagent kit for performing the above-described antibody assay.

The kit contains the above-mentioned antibody assay device as an essential component, and is used for diluting various reagents such as a sample diluent as described above, as well as a dropper and a test sample. Accessories such as ampules (tubes) may be included.

The present invention includes the following aspects: (1) at least (a) a first region to be brought into contact with a test sample and (b) a second region to react with an antibody in the test sample. An assay device comprising a solid support provided so that a test sample is transported from the first region to the second region by capillary action, and a labeling means for detecting a reaction result in the second region And (b) immobilizing, on the second region (b), a test site in which (i) a ligand for capturing a target antibody to be assayed is immobilized, and (ii) a ligand for capturing any antibody in a test sample. And (2) an antibody assay device according to (1), wherein the ligand immobilized on the test site is an antigen against a target antibody in a test sample. ) Reagents immobilized on the control site The antibody assay device according to (1) or (2), wherein Gand is an anti-human immunoglobulin antibody that captures any antibody in the test sample: (4) As a labeling means, any of a target antibody and any antibody The antibody assay device according to any one of (1) to (3), which has a labeled ligand to be bound: (5) the labeling means is a labeled ligand that binds to both a target antibody and an arbitrary antibody; The labeled ligand is detachably supported in a region upstream of the second region of the solid support, and when contacted with a test sample, reacts with a target antibody and any antibody in the sample to react with the target antibody. Forming an antibody / labeled ligand-conjugate and an optional antibody / labeled ligand-conjugate, which are transported to the second region by capillary action and bind to the respective test and control sites; 4) antibody assay device according.

(6) The antibody assay device according to (5), wherein the labeled ligand is supported on a region (tracer region) located between the first and second regions of the solid support. The antibody assay device according to any one of (4) to (6), wherein the labeled ligand that binds to both the target antibody and any antibody is a labeled anti-human immunoglobulin antibody. The antibody assay device according to (7), wherein the human immunoglobulin antibody is an anti-IgG antibody having binding specificity to the Fc region of immunoglobulin G: (9) the solid support is in the first region and the second region. The antibody according to any one of (1) to (8), further comprising an absorption region, whereby the test sample transported from the first region to the second region is further transported to the absorption region by capillary action. Assay device: ( 10) The antibody assay device according to any one of (1) to (9), wherein the binding reaction of the target antibody at the test site in the second region is performed in the presence of a component derived from Escherichia coli.

(11) The test sample is urine (1) to (1)
The antibody assay device according to any one of (0): (12) An antibody assay kit including the antibody assay device according to any one of (1) to (11): (13) The test sample is prepared by using (1) to (9). Contacting the first region of the antibody assay device according to any one of the above, and detecting the presence or absence of color development of the test region in the second region under the condition that the control region in the second region is colored. The solid-phase assay method for the target antibody in the test sample: (14) The solid-phase assay method for the target antibody according to (13), wherein the test sample is urine: (15) At the test site in the second region of the antibody assay device The solid-phase assay method for a target antibody according to (13) or (14), wherein the binding reaction of the target antibody is performed in the presence of a component derived from Escherichia coli.

[0087]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be specifically described below with reference to the following embodiments. The following examples describe each component of the antibody assay device of the present invention, a preferred method of manufacturing and using the device utilizing the same, and an assay procedure using the same. However, these examples are only one embodiment of the present invention, and the present invention is not limited to these examples.

Example 1 (1) H. Preparation of H. pylori Antigen Culture of H. pylori (clinical isolate) in Brucella agar medium (Becton) for 48 hours (10% CO ₂ , 5% O ₂ , 37 ° C)
The cells were collected with cold PBS. After five centrifugal washes with cold PBS,
Add cold PBS so that the cell concentration is 100 mg / ml, and add an equal volume of cold 0.2% Triton X-100 under stirring.
The BS solution was added. After stirring for 5 minutes and centrifuging, the supernatant obtained was A H. pylori antigen solution was stored at -80 ° C.

(2) Preparation of Dry Glass Fiber Containing Labeled Anti-Human IgG Antibody A colloidal gold-labeled anti-human IgG (Fc-specific) having a diameter of 40 nm was placed on a glass fiber sheet (5.0 mm × 260 mm, 0.8 mm thick, manufactured by Whatman). Target) 1 ml of antibody solution was added and dried overnight to prepare. It was stored at room temperature with desiccant until use.

(3) Preparation of membrane H. pylori antigen solution (3 mg / m
l) and an anti-human IgG antibody (0.3 mg / ml) were each applied to a nitrocellulose membrane (26.5 mm x 260 m).
m, thickness 0.1 mm; manufactured by Advance Micro Devices Co., Ltd.) and sprayed (1.5 μl / cm) into lines drawn at regular intervals as shown in FIG.
Dry for 0 minutes. After drying, the plate was washed by immersing it in Borax buffer (pH 8.2) containing skim milk for 30 minutes. It was dried at 37 ° C. for 1 hour and stored with a desiccant at room temperature.

(5) Assembly (Solid Support) As shown in FIG. 3A, the above-mentioned membrane (3) and water-absorbing filter paper pad (4) (22 × 260 mm, thickness 1.5 m)
m; manufactured by Whatman), the glass fiber (2) containing the labeled antibody and the sample pad (1) (15 × 260 m
m, thickness: 1.0 mm; filter paper, manufactured by Whatman) with an adhesive, and cut with a width of 5 mm.

This is placed in the lower section (8) of the molded plastic housing as shown in FIG. 3 (B), and then the two openings in which the sample addition window (9) and the detection window (10) are arranged. The upper section (7) of the housing having the above was overlaid on the support and integrated with the lower section (8).

Example 2 Using an apparatus prepared in Measurement Example 1 pyrophosphoric Li urinary antibody, H. H. pylori infection in three types of urine specimens: urine of the infected person, uninfected urine, and extremely thin urine of the infected person. H. pylori antibodies were measured.

First, a sample diluent (200 mM Tris, 0.14 M Na
Cl, 2% casein, 0.5% BSA, 0.05% Tween 20, 0.1% NaN
₃ (pH 7.3), Escherichia coli LPS (manufactured by Gibco) 50 μg / m
l) 500 μl of a urine sample was added to 500 μl and mixed, and 6 drops (about 150 μl) of the mixed solution were dropped into the sample addition window (9) of the apparatus described in Example 1 and allowed to adsorb to the support.
Let stand for a minute. As a result, when a suitable urine sample is used as a subject, a pink to red coloring band appears at a control site of the detection window (10), and when an extremely thin unsuitable urine sample is used as a subject, The test site and the control site in the detection window (10) did not develop any color, indicating an undeterminable result. When a suitable urine sample was used,
When the cells are not infected with H. pylori, a pink to red color band appears only at the control site of the detection window (10),
H. A negative result (true negative) for H. pylori infection;
H. In the case of infection with H. pylori, pink to red color bands appear in both the test site and the control site in the detection window (10). He showed a positive result for H. pylori infection.

Example 3 Except using Escherichia coli-derived ingredient prepared by the following method instead of the pyro Li urine measuring antibodies (1) E. coli LPS formulated in sample dilution buffer of Example 2, in the same manner as in Example 2 H. The measurement of H. pylori urinary antibody was performed. As a result, the same result as in Example 2 was obtained. m (2) Preparation of Escherichia coli-Derived Components Escherichia coli (pvc18 / JM109 strain: manufactured by Takara Shuzo) was converted to ampicillin-containing liquid LB medium (Luria-Bertani medium, Nippon Pharmaceutical)
After culturing at 37 ° C. for 18 hours, the cells were collected by centrifugation, and PBS was collected.
And washed twice. Cold PBS was added so that the cell concentration was 100 mg / ml, and the mixture was crushed and extracted with an ultrasonic crusher (10 seconds × 3 times). The centrifuged supernatant thus obtained was used as an E. coli extracted protein.

Example 4 The most accurate H. of the existing diagnostic methods ¹³ C-UBT test [J. Gastroenterol.,
33: 6-13 (1998)]. Whole blood and plasma of 21 cases (42 cases in total) of H. pylori infection positive and negative individuals
Urine was used as a test sample, and H.U. H. pylori antibodies were measured according to Example 2 above.

[0097] As a comparative experiment, a commercially available H.V. Using a H. pylori antibody measurement device,
The measurement was performed on a sample of the same subject, and the effect of the apparatus of the present invention was examined from the result.

FIG. 5 shows the results.

In the figure, the comparison devices A to H indicate the following measurement devices.

A: Helitest (Cortecs Diagnostics) B: H.pylori-Check-1 (Bio-Medical Products) C: First Check H.pylori (Worldwide Medical Corp.)
D: Biocard Helicobacter pylori IgG (Anti Biotech
E: Insta Test H. Pylori (Cortez Diagnostics Inc.) F: One Step H. pylori Test (Teco Diagnostics) G: H. pylori SPOT (International Immuno-Diagnostic)
H: Quick Stripe H.pylori (Diatech Diagnostics In)
c. companies, Ltd.) In the figure "Specifity" is the ¹³ C-UBT test-negative samples when measured with each kit, shows the percentage could be detected as negative (negative rate), "Sensitivity" will, ¹³ C
-Indicates the percentage (positive rate) of UBT test positive samples that were detected as positive when measured with each kit.

As can be seen from the results shown in FIG. 5, the assay device and assay method of the present invention showed that blood (whole blood, plasma,
It can be seen that this is an excellent assay system having high detection specificity and accuracy, not only when serum is used but also when urine is used.

From the results, it can be said that according to the present invention, even if safe and simple urine is used as a sample, highly sensitive and highly specific antibody detection is possible, which is useful in the field of clinical testing. .

[0103]

[Experimental example] Experimental example 1 For measurement of urinary antibody of E. coli-derived components
Effects on (1) The effects of E. coli-derived components on the measurement of urinary antibodies were evaluated using the measurement system of Example 2. Specifically, ¹³ C
H. grouped by the UBT test. Urine of H. pylori infection-positive and -negative individuals were used as test samples, respectively. H. H. pylori antibodies were measured. The color of the lines of the test site and the control site is determined by a densitometer (ATTO
(Manufactured by the company).

Table 1 shows the results.

[0105]

[Table 1]

E. coli LPS was added to the test sample at 11.1 μg /
It was found that the addition of at least ml eliminates the non-specific reaction observed in Diluent 1 and prevents false positive detection (erroneous detection).

(2) Next, an experiment was conducted to confirm that the incorporation of E. coli LPS into the test sample (reaction system) did not affect the line strength. Specifically, ¹³ CU
A system not containing Escherichia coli LPS was used for a total of 8 BT test positive urine samples and 4 negative urea urine samples (a negative urine sample that did not show a false positive reaction in the test of (1) was used in advance). (Diluent 2) and E. coli LPS
Using a buffer containing 50 μg / ml, the reactivity of the antibody in urine was examined according to the method of Example 2. Table 2 shows the results.

[0108]

[Table 2]

From these results, it was found that LPS blended in the reaction system
Did not affect the line strength.

Experimental Example 2 Known method [Calypte ^™ HIV-1 Urine EIA: Arch. Patho
l. Lab. Med., 119, 139-141 (1995); Clinical lnfec.
Tious Diseases, 19, 1100-1104 (1994)]
Using a urine sample that gave a false positive result in the measurement of an IV antibody, a study was conducted to identify components that give a nonspecific reaction in the measurement system.

(1) The above urine sample was adjusted to pH 7.4 with 1M phosphate buffer (pH 7.7), and then adjusted to 5.0, 0.
Filtration was carried out sequentially with an 8 and 0.2 μm cut filter, and 20 ml of the filtrate was concentrated to 2 ml by ultrafiltration (10 kd cut membrane). The concentrated urine sample was subjected to gel filtration (Sephacryl S-300, Pharmacia), and each of the obtained fractions was tested for reactivity with HIV antigen.

Incidentally, the reactivity to the HIV antigen was determined by dividing each fraction into H
After reacting with the HIV antigen-immobilized plate on which the IV antigen was immobilized, the binding component (antigen-antibody conjugate) was converted to an ALP-labeled goat anti-human (IgG + IgM) antibody (Jackson ImmunoRese).
arch Labs.). As a result of this test, the presence of non-specific reaction components peaking at fractions 46 to 48 was confirmed.

Similarly, the component bound to the antigen-immobilized plate was converted to an ALP-labeled goat anti-human IgG (Fc-specific).
Antibody or HRP-labeled goat anti-human IgG (Fab specific)
Detection was performed using an antibody (both manufactured by Jackson ImmunoResearch Labs.). FIG. 6 shows the results.

In FIG. 6, the vertical axis represents absorbance (OD),
The horizontal axis indicates the fraction (fraction number) of gel filtration. The solid line is the absorbance of the protein at 280 nm, the black circle is the detection result with the anti-human (IgG + IgM) antibody, the white triangle is the detection result with the anti-human IgG (Fc-specific) antibody, and the black triangle is the anti-human The results of detection using an IgG (Fab-specific) antibody are shown.

From the figure, in the detection with an anti-human IgG (Fab-specific) antibody, the same reaction pattern (reactivity with non-specific reaction components) as in the case of the anti-human (IgG + IgM) antibody was shown. It was revealed that no reactivity was exhibited when a human IgG (Fc-specific) antibody was used. From this, it was considered that the component giving the non-specific reaction was a fragmented or denatured human IgG retaining the reactivity with an anti-human IgG (Fab-specific) antibody.

(2) Each of the above fractions was subjected to goat anti-human IgG
(H + L) antibody (Jackson ImmunoResearch Labs.)
With HRP-labeled goat anti-human IgG
(Fc-specific) antibody, HRP-labeled goat anti-human IgG (F
The amount of each globulin was measured by reacting with an ab-specific) antibody or an HRP-labeled rat anti-human IgM antibody (both manufactured by Jackson ImmunoResearch Labs.).

As a result, the IgG (Fc) concentration was lower than the detection sensitivity near the fraction 50 with the peak of the fraction 38, whereas the IgG (Fab) concentration was equivalent to that of the IgG (Fc) up to the fraction 46. Concentration, and thereafter showed only a mild decreasing trend. In particular, the difference was remarkable after fraction 47. This implies that up to fraction 46, both measurements reflect the concentration for intact IgG, whereas the IgG (Fab) concentration for fraction 47 and onward shows that anti-human IgG (Fab specific) This is considered to reflect the concentration of the fragmented IgG or the denatured IgG retaining the reactivity with the antibody.

[0118] IgM was not detected in any of the fractions measured.

(3) As described above, HR of fraction 38 considered to be rich in complete IgG and fraction 47 considered to be rich in non-specific reaction components were determined.
P-labeled goat anti-human IgG (Fc-specific) antibody or HRP
Western blot analysis using a labeled goat anti-human IgG (Fab specific) antibody was performed.

As a result, in fraction 38,
The results of analysis using both labeled antibodies were both human IgG (control).
The reaction pattern was very similar to that of the above. Fraction 47
In the analysis with a human IgG (Fc-specific) antibody, a main spot of human IgG and a spot slightly reacting with 43 KD were observed, and this 43 KD spot was a heavy chain (H chain) fragmented from its molecular weight. I thought there was. In the analysis of the fraction 47 using a human IgG (Fab-specific) antibody, spots not detected by human IgG were observed at around 20 and 40 KD, and it was considered that components giving a non-specific reaction corresponded to these spots.

From the above, it is considered that the component that gives a non-specific reaction when measuring urinary HIV antibody is a fragmented or denatured IgG that retains reactivity with an anti-human IgG (Fab-specific) antibody. And from its molecular weight,
It was considered to be a substance involved in the L chain such as an IgG light chain (L chain) and a dimer thereof.

Thus, the anti-human IgG (Fc), which has no reactivity with a component that gives a non-specific reaction in the antibody assay system (assay apparatus, assay method) of the present invention.
By using the (specific) antibody as an antibody detection reagent (labeling means in the present invention, ie, a labeled ligand which is immobilized on the tracer region of the solid support), non-specific reactions can be prevented in antibody detection, Thus, it has been clarified that a reduction in false positive detection enables high-precision antibody detection with high specificity.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a strip-shaped solid support included in the assay device of the present invention.

FIG. 2 is a diagram showing the principle of measuring a target antibody in a test sample by the assay device of the present invention.

FIG. 3 is a diagram showing an example of the assay device of the present invention.

FIG. 4 is a diagram showing a test site and a control site in a second area (Example 1 (3)).

FIG. 5 is a diagram showing the results of Example 4.

FIG. 6 is a graph showing gel filtration of a urine sample giving a false positive result in the measurement of urinary HIV antibody, and the antibody reactivity of each fraction. In the figure, the vertical axis indicates absorbance (OD), and the horizontal axis indicates gel filtration fractions (fraction numbers). Solid line is 28
The absorbance of the protein at 0 nm, the solid circles indicate the results of detection with the anti-human (IgG + IgM) antibody, the open triangles indicate the results of detection with the anti-human IgG (Fc-specific) antibody, and the solid triangles indicate the anti-human IgG ( (Fab specific) The detection result by an antibody is each shown.

[Explanation of symbols]

 1. First area 2. Tracer area 3. Second area 4. Third region 5. Test site Control site 7. 7. Upper section of housing 8. Lower section of housing Sample addition window 10. Detection window

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Machikawa Fubo, 19-18 Inui, Sumiyoshi, Aizumi-cho, Itano-gun, Tokushima Prefecture (72) Inventor Shigeo Takahashi Inventor Tetsuya Tachikawa 13-6 Higashino Kamimoto, Takabo, Kitajima-cho, Itano-gun, Tokushima

Claims (1)

[Claims] At least a first step of contacting a test sample
A solid support formed by providing a region and (b) a second region for reacting an antibody in the test sample such that the test sample is transported from the first region to the second region by capillary action; and What is claimed is: 1. An assay device comprising a labeling means for detecting a reaction result in a second region, wherein the (b) a ligand immobilizing a target antibody to be assayed is immobilized on the second region. An antibody assay device comprising: a site; and (ii) a control site on which a ligand for capturing any antibody in the test sample is immobilized.