JP7153545B2 - Immunochromatographic test strip, test substance measurement method and immunochromatographic test kit using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunochromatographic test strip, a test substance measuring method using the same, and an immunochromatographic test kit, and more specifically, an immunochromatographic test strip comprising a test line and a control line, and a test substance measuring method using the same. and immunochromatographic test kits.

In order to make a positive/negative determination of an infectious disease and to identify its cause, it is necessary to detect pathogens such as bacteria and viruses in samples collected from subjects. It is also known to detect and quantify a substance that serves as a marker for a specific disease from a sample such as blood for positive/negative determination of the disease. Examples of the method for detecting the pathogen include a genetic testing method for detecting genes characteristic of the pathogen from a sample, a method for identifying the pathogen by electron microscopic observation, and when the pathogen is a bacterium, culture for identification by isolating and culturing it. laws are known. Immunological test methods such as ELISA and IFA using antigen-antibody reaction are known as methods for detecting and quantifying the above pathogens and markers. However, these measurement methods require cumbersome sample processing, require skilled techniques for measurement, require special equipment, take a long time to obtain results, and require a large number of samples in a short period of time. There is a problem that it is not possible to test the specimen of

On the other hand, immunochromatography, which is another immunological testing method, is known as a simple and rapid measurement method. As the immunochromatographic method, for example, a sample is developed from one end of an immunochromatographic test strip consisting of a porous membrane on which a substance (such as an antigen or antibody) that can specifically bind to the test substance in the sample is immobilized. , in the course of its development, a labeling reagent capable of specifically binding to the test substance (a substance that is specifically capable of binding to the test substance and is labeled (such as an antigen or antibody)) and the test substance form a complex with, indirectly or directly capture the complex with the antigen or antibody immobilized on the porous membrane, and measure the test substance. In such a method, a sample such as blood, urine, or feces is applied or dropped onto the immunochromatographic test piece (if necessary, the sample is suspended in a pretreatment liquid or filtered with a filtration filter), and the porous Since the test substance in the sample can be measured simply by spreading the sample, the complex, etc. on the membrane by capillary action, the operation is simple, and visual judgment is also possible. Widely used.

In general immunochromatographic test strips, a test line in which the complex is indirectly or directly captured by the antigen or antibody immobilized on the porous membrane and detected as a signal, and whether or not the test is established As an indicator for determination, a control line on which a substance (control reagent) that specifically binds to the labeling reagent but does not bind to the test substance is immobilized is provided. When a signal is detected in the control line when the sample is developed from one end or one point of the immunochromatographic test strip, the amount of sample and labeling reagent required for measurement is developed without problems on the test line and control line. It means that each antigen-antibody reaction etc. was performed normally on the immunochromatographic test piece, but if no signal is detected in the control line, the amount of sample and / or labeling reagent required for measurement is not developed, and each antigen-antibody reaction may not be performed normally, so the test is considered unsuccessful. As described above, the control line has a great influence on the success or failure of the test, so it is desired that the control line always shows stable signal intensity.

Antibodies against labeling reagents are generally used as control reagents immobilized on the control line. For example, when a rabbit monoclonal antibody specific to a test substance is used as the labeling reagent, an anti-rabbit antibody is used as the control reagent.

Biological samples such as blood sometimes contain specimens containing heterophile antibodies against various animal antibodies, such as human anti-mouse antibody (HAMA) and human anti-rabbit antibody (HARA). Therefore, for example, if a specimen containing a heterophilic antibody against an animal of the same species as the animal used to prepare the antibody constituting the labeling reagent is tested, a non-specific reaction will occur. For example, when a rabbit monoclonal antibody specific to the test substance is used as the labeling reagent and the antibody immobilized on the porous membrane, and a specimen containing HARA is used as a sample, HARA reacts with these rabbit monoclonal antibodies. This can lead to false-positive results as if the test substance was present even in samples that did not contain the test substance. Such non-specific reactions impair the advantage of the immunochromatographic method, which is the ability to measure trace components specifically and with high sensitivity.

Various attempts have been made to suppress the non-specific reaction caused by the heterophilic antibody and to obtain correct measurement values. For example, Japanese Patent Application Laid-Open No. 61-065162 (Patent Document 1) discloses that a mouse-derived substance such as mouse serum is present as a non-specific reaction inhibitor in an immunoassay method using a mouse-derived monoclonal antibody. is described. However, in the immunochromatographic method, for example, when a mouse monoclonal antibody specific to the test substance is used as the labeling reagent and an anti-mouse antibody against it is used as the control reagent, the nonspecific reaction inhibitor is mouse-derived When the substance is used, the control reagent captures the non-specific reaction inhibitor, inhibiting the capture of the labeling reagent to the control reagent (control line), thereby significantly reducing the signal intensity of the control line. I had a problem.

Attempts have also been made to reduce the effects of interfering substances including heterophilic antibodies such as HAMA and HARA. is a method for quantifying the concentration of a test substance (test substance) in a fluid sample, comprising a step of detecting a signal from a detectable labeled substance corresponding to the concentration of the test substance; detecting a signal (internal control signal) from a labeled substance that is detectable without dependence on the concentration of the substance; Methods are described that use combinations of ligand pairs that do not substantially undergo , such as biotin or biotin analogues and avidin or streptavidin, or haptens and antibodies. In addition, Japanese Patent Application Laid-Open No. 2009-085751 (Patent Document 3) discloses that, as an immunochromatographic test device, a substance capable of reacting with a human anti-mouse antibody (HAMA) is immobilized with a binding substance capable of binding to a component to be detected. The document describes a test piece arranged upstream in the direction of sample development from the judgment part, and in the examples of the document, latex particles sensitized with a labeling substance for control and streptavidin as a substance capable of binding thereto. and biotinylated BSA have been described. However, in these methods and test strips, in order to detect a signal in the control line, a combination of ligand pairs (Patent Document 2) or a control that is substantially unaffected by substances coexisting in the test sample is used. Therefore, it is necessary to separately prepare a labeling substance (Patent Document 3), which complicates the preparation of reagents and test strips and increases the cost.

Further, for example, Japanese Patent Application Laid-Open No. 2013-205335 (Patent Document 4) describes, as an immunochromatographic test device, a first affinity labeling reagent that specifically binds to a test substance via avidin and biotin. A test strip is described in which a substance and a labeling substance are linked and the control reagent contains biotin. However, such a test strip also has the problem that the preparation of the labeling reagent is complicated.

Furthermore, for example, International Publication No. WO 2017/065213 (Patent Document 5) discloses, as an immunochromatographic test strip, a detection reagent that specifically binds to a substance to be detected (test substance) and a conjugation impregnated with a control line detection reagent. A test strip having a pad (conjugate pad) and a porous membrane pad on which an antibody for specifically capturing the control line detection reagent is immobilized is described, and the control line detection reagent is biotin-labeled. The use of protein-bound detection particles and the use of an anti-biotin antibody as an antibody to specifically capture the control line detection reagent is described. However, even with such a test strip, it is necessary to prepare a separate detection reagent (control line detection reagent) in the same manner as the methods and test strips described in Patent Document 2 and Patent Document 3, and the preparation of reagents and test strips is complicated. However, the problem was the increase in cost.

JP-A-61-065162 JP 2001-033454 A JP 2009-085751 A JP 2013-205335 A WO2017/065213

When the present inventors conducted further studies on immunochromatographic test strips, in addition to the above problems, in conventional immunochromatographic test strips, for example, separate detection as described in Patent Documents 2, 3, and 5 When using reagents, since these are particles, the presence of such separate particles may cause clogging of the porous membrane, increase the background, reduce the detection accuracy of the test substance, and It has been found that the signal in the control line is an indirect evaluation by the other particles, so there is a problem that the accuracy of determining whether the test is successful or not may be lowered.

The present invention has been made in view of the above problems of the prior art, and in the immunochromatographic method, there is no need to separately prepare reagents other than labeling reagents and control reagents in order to detect signals in the control line, and , an immunochromatographic test strip with a control line signal formation mechanism that does not affect the signal intensity in the control line even if a non-specific reaction inhibitor against heterophilic antibodies is present in the sample, and a test substance using the same An object of the present invention is to provide a measurement method and an immunochromatography test kit.

The present inventors have made intensive studies to achieve the above object, and as a result, have found that, as the labeling reagent, a labeled substance and a first compound that is bound to the labeled substance and is capable of specifically binding to the test substance In order to suppress non-specific binding of the label to substances other than the first substance, using a labeling reagent that is provided with one substance and blocked with a blocking agent, and conventionally By newly adopting a third substance that can specifically bind to the blocking agent that has been used as the control reagent and immobilizing it on the control line, It was found that a sufficiently strong signal could be detected in the control line without separately preparing a reagent. Furthermore, even when a non-specific reaction inhibitor derived from each species is used, the third substance does not capture it, and the signal intensity in the control line is not affected. The inventors have found that the use of an appropriate amount of a reaction inhibitor can prevent false positive determination by heterophilic antibodies, and have completed the present invention.

The aspects of the present invention obtained from such findings are as follows.
[1] An immunochromatographic test strip for measuring a test substance in a sample,
Equipped with conjugate pads, test lines, and control lines,
The conjugate pad retains an elutable labeling reagent containing a first substance capable of specifically binding to the test substance, a blocking agent, and a label,
the label is bound to the first substance and blocked with the blocking agent;
A second substance that can specifically bind to the test substance is immobilized on the test line,
A third substance that can specifically bind to the blocking agent is immobilized on the control line,
An immunochromatographic test strip characterized by:
[2] In [1], wherein the blocking agent is at least one protein selected from the group consisting of casein, E. coli-derived heat shock protein, bovine serum albumin, human serum albumin, and skim milk. Immunochromatographic test strip as described.
[3] The sample contains a non-specific reaction inhibitor, or the conjugate pad holds the non-specific reaction inhibitor in a further elutable manner, [1] or [2] ] The immunochromatographic test strip described in .
[4] The non-specific reaction inhibitor is selected from the group consisting of normal mouse globulin, normal rabbit globulin, normal bovine globulin, normal goat globulin, normal sheep globulin, normal rat globulin, normal alpaca globulin, and normal chicken globulin. The immunochromatographic test strip according to [3], which is at least one.
[5] The third substance is at least one organism-derived anti-blocking agent antibody selected from the group consisting of mouse, rabbit, cow, goat, sheep, rat, alpaca, and chicken. The immunochromatographic test strip according to any one of [1] to [4].
[6] A method for measuring a test substance in a sample using the immunochromatographic test strip according to any one of [1] to [5],
contacting the sample with the conjugate pad to form a complex between the test substance in the sample and the labeling reagent;
Capturing and detecting the complex via the test substance with the second substance in the test line;
Capturing the labeling reagent with the third substance through the blocking agent in the control line;
A method for measuring a test substance, comprising:
[7] Further comprising the step of adding a non-specific reaction inhibitor to the sample or the conjugate pad, or the conjugate pad holds the non-specific reaction inhibitor so that it can be further eluted. The test substance measurement method according to [6].
[8] An immunochromatographic test kit comprising the immunochromatographic test strip according to any one of [1] to [5].
[9] The immunochromatographic test kit of [8], further comprising a non-specific reaction inhibitor.
[10] The non-specific reaction inhibitor is selected from the group consisting of normal mouse globulin, normal rabbit globulin, normal bovine globulin, normal goat globulin, normal sheep globulin, normal rat globulin, normal alpaca globulin, and normal chicken globulin. The immunochromatography test kit according to [9], characterized by comprising at least one.

According to the present invention, in the immunochromatographic method, there is no need to separately prepare reagents other than the labeling reagent and the control reagent in order to detect the signal in the control line, and non-specific reaction suppression against heterophile antibodies in the sample It is possible to provide an immunochromatographic test strip with a control line signal formation mechanism that does not affect the signal intensity in the control line even in the presence of an agent, and a method for measuring a test substance and an immunochromatographic test kit using the same. becomes.

According to the present invention, since there is no need to separately prepare reagents other than labeling reagents and control reagents, immunochromatographic test strips equipped with a control line exhibiting stable signal intensity can be produced simply and at low cost. becomes possible. In addition, since the control line is not affected by the non-specific reaction inhibitor, an appropriate amount of the non-specific reaction inhibitor can be used, making it possible to avoid the influence of heterophile antibodies in the sample. Therefore, it is possible to increase the certainty of determination on the test line and the control line.

1 is a schematic perspective view of an immunochromatographic test strip according to one embodiment of the present invention; FIG.

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in detail with reference to its preferred embodiments.

The immunochromatographic test strip of the present invention is an immunochromatographic test strip for measuring a test substance in a sample, and comprises at least the following conjugate pad, test line and control line.

Further, the method of measuring a test substance of the present invention is a method of measuring a test substance in a sample using the immunochromatographic test strip of the present invention, and the immunochromatographic test kit of the present invention comprises at least the immunochromatographic test strip of the present invention. A kit comprising an immunochromatographic test strip.

(Test substance)
In the present invention, the "test substance" to be measured is not particularly limited, and includes substances such as pathogens or sugar chains derived from them, substances that serve as markers for specific diseases, and more specifically, bacteria , protists and fungi, viruses, proteins, polysaccharides, nucleic acids, etc., such as Legionella, Streptococcus pneumoniae, Group A β-hemolytic streptococcus, Norovirus (NV), Rotavirus, Adenovirus, RS virus (RSV ), influenza virus, etc., but are not limited to these.

(sample)
In the present invention, the "sample" used for measuring the test substance is not particularly limited as long as it is a sample in which the test substance can be present. Examples of the sample include, but are limited to, urine, feces, sputum, throat swab, blood, serum, plasma, saliva, ear discharge, and the like collected from a subject (preferably human). is not. In this specification, "specimen" is also used synonymously with "sample". In addition, for example, in immunochromatographic test strips using biotin as used in Patent Documents 4 and 5, the signal intensity of the control line is significantly attenuated when a biotin-containing specimen such as blood or urine is used as a sample. However, since it is not necessary to use biotin in the present invention, specimens such as blood and urine containing biotin can also be suitably used as samples.

The sample is preferably a fluid sample, more preferably an aqueous solution or aqueous dispersion. good too.

The pretreatment liquid includes a solution for pretreating or preserving the sample, such as an extraction solution, a dilution solution, a suspension solution, a preservation solution, and the like. Such a pretreatment liquid preferably contains a buffer consisting of a buffer and a solvent (preferably water). As the buffering agent, a buffering agent that can be adjusted to a pH suitable for the desired immune reaction is used. In general, the pH of the buffer solution is preferably 5 to 10. In this case, buffers such as phosphate buffer, Tris buffer, Good's buffer, borate buffer and the like can be used. A more suitable pH of the buffer solution is preferably 6 to 8. In this case, for example, Good's buffer solution such as HEPES or PIPES is preferable.

In addition to the buffering agent, sodium chloride or the like may be further added to the pretreatment liquid, or a known additive may be further added. Examples of the additives include proteins whose main purpose is to promote antigen-antibody reactions or suppress non-specific reactions (e.g., free antibodies, bovine serum albumin, casein, gelatin, etc.), polymeric compounds (e.g., polyethylene glycol, dextran, methylcellulose, polyvinylpyrrolidone, etc.), nonionic surfactants (eg, Tween (registered trademark) 20, Triton (registered trademark) X-100, etc.), ionic surfactants, polyanions (eg, dextran sulfate , heparin, polystyrene sulfonic acid, hyaluronic acid, chondroitin sulfate, etc.) or salts thereof; sodium azide and long-chain alkyl quaternary ammonium salts as antibacterial agents; One of them may be used alone, or two or more of them may be used in combination.

Moreover, as the sample according to the present invention, a sample to which a non-specific reaction inhibitor is further added may be used. According to the present invention, even if a non-specific reaction inhibitor is added to the reaction system, the signal intensity in the control line can be maintained at a sufficient level without being affected by it. In the present invention, "non-specific reaction inhibitor" means human anti-mouse antibody (HAMA), human anti-rabbit antibody (HARA), human anti-bovine antibody, human anti-goat antibody (HAGA), human anti-sheep antibody (HASA). , human anti-rat antibodies, human anti-alpaca antibodies, human anti-chicken antibodies, etc., which are human antibodies that recognize antibodies derived from various organisms. The non-specific reaction inhibitor binds to the heterophilic antibody, that is, by absorbing the reactivity of the heterophilic antibody, the heterophilic antibody is the following first substance and / or second substance to prevent false positive determinations from occurring. Examples of such a non-specific reaction inhibitor include biological antibodies that can be recognized and bound by the heterophilic antibody. One of these may be used alone or two or more may be used in combination. may For example, non-specific reaction inhibitors against HAMA include mouse antibodies (Normal Mouse Globulin (NMG), HAMA Blocker, etc.), and non-specific reaction inhibitors against HARA include rabbit antibodies (Normal Rabbit Globulin (NMG), HAMA Blocker, etc.). Globulin (NRG)), etc.), bovine antibodies (normal bovine globulin (NBG), etc.) as non-specific reaction inhibitors against human anti-bovine antibodies, goat antibodies (Normal Bovine Globulin (NBG), etc.) as non-specific reaction inhibitors against HAGA Normal goat globulin (NGG), etc.), sheep antibody (Normal Sheep Globulin (NSG), etc.) as a non-specific reaction inhibitor against HASA, non-specific reaction against human anti-rat antibody Rat antibodies (Normal Rat Globulin (NRG), etc.) as inhibitors, and alpaca antibodies (Normal Alpaca Globulin (NAG), etc.) as non-specific reaction inhibitors for human anti-alpaca antibodies. However, non-specific reaction inhibitors against human anti-chicken antibodies include chicken antibodies (Normal Chicken Globulin (NCG)), etc., and serum containing at least one of these When the non-specific reaction inhibitor is added to the sample, the type and amount thereof can be appropriately adjusted depending on the type and concentration of the sample to be measured.

(measurement)
In the present invention, "measurement of the test substance" includes detection to confirm the presence or absence of the test substance, and quantification or semi-quantification of the amount of the test substance. In the present invention, the measurement of the test substance is performed by detecting the signal in the test line and optionally quantifying it, and the success or failure of the measurement is determined by detecting the signal in the control line and optionally The above-mentioned "signal" includes coloration (color development), reflected light, luminescence, fluorescence, radiation from radioactive isotopes, etc., and in addition to those that can be confirmed with the naked eye, signals It also includes those that can be confirmed by detection methods and devices according to the type.

<Immunochromatographic test piece>
The immunochromatographic test strip of the present invention is
An immunochromatographic test strip for measuring a test substance in a sample,
Equipped with conjugate pads, test lines, and control lines,
The conjugate pad retains an elutable labeling reagent containing a first substance capable of specifically binding to the test substance, a blocking agent, and a label,
the label is bound to the first substance and blocked with the blocking agent;
A second substance that can specifically bind to the test substance is immobilized on the test line,
A third substance that can specifically bind to the blocking agent is immobilized on the control line,
It is characterized by

In addition, as the immunochromatographic test strip of the present invention,
a sample pad in contact with the conjugate pad; and a porous membrane in contact with the conjugate pad and having the test line and the control line;
is preferably further provided.

Preferred embodiments of the immunochromatographic test strip of the present invention will be described below in detail by taking one embodiment of the present invention as an example with reference to the drawings, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are denoted by the same reference numerals, and overlapping descriptions are omitted.

FIG. 1 is a perspective view showing a schematic diagram of one embodiment (test strip 100) of the immunochromatographic test strip of the present invention. The test strip 100 is a lateral flow immunochromatographic test strip, and is porous with a sample pad 140 for receiving a sample, a conjugate pad 130 holding a labeling reagent in an elutable manner, a test line 122 and a control line 124. a membrane 120; In addition, the immunochromatographic test strip of the present invention may further comprise an absorbent pad 150 and a sheet 110 (for example, a plastic adhesive sheet) supporting them, as shown in FIG. 1, if necessary. Furthermore, the immunochromatographic test strip of the present invention may comprise a casing (not shown) containing a series of sample pads, conjugate pads, porous membranes, absorbent pads, sheets, etc., said casing comprising: Windows may be provided for recognizing sample pads, conjugate pads, test lines, control lines, and the like. The size and spacing of these members can be appropriately adjusted according to the type of test substance, purpose of measurement, and the like. In the description referring to FIG. 1 below, the sample pad 140 side of the porous membrane 120 is referred to as upstream (upstream in the direction of sample development), and the absorbent pad 150 side is referred to as downstream (downstream in the direction of sample development). do.

In FIG. 1, the conjugate pad 130, the test line 122, and the control line 124 are arranged in this order from upstream, but the arrangement of the immunochromatographic test strip of the present invention is not limited to this, and the present invention The test line and the control line may be arranged at the same distance from the conjugate pad. For example, the test line and the control line may be arranged concentrically around the conjugate pad. Alternatively, bar-shaped test lines and control lines may be arranged to intersect each other to form a plus (+) symbol or the like. Also, the test line according to the present invention may be arranged downstream of the control line according to the present invention.

(sample pad)
A sample pad according to the present invention (sample pad 140 in FIG. 1) functions as a sample supply unit that receives a dropped or applied sample. Furthermore, the sample pad can also have a function of filtering insoluble particles in the sample and a function of preventing the sample from overflowing. The material of the sample pad is preferably a material having uniform properties, such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, non-woven fabric, and cotton cloth. The thickness and size of the sample pad are not particularly limited and can be adjusted as appropriate.

(Conjugate pad)
The conjugate pad according to the present invention (the conjugate pad 130 in FIG. 1) functions as a labeling reagent holding portion that holds the labeling reagent in an elutable manner. The material of the conjugate pad is preferably a porous material such as cellulose filter paper, glass fiber, and non-woven fabric, among which glass fiber is more preferred. The thickness of the conjugate pad is not particularly limited, and is usually 260 to 950 μm. The area of the conjugate pad is also not particularly limited, and is usually 10 to 100 mm ² .

[Labeling reagent]
In the present invention, the labeling reagent held by the conjugate pad includes a label, a first substance capable of specifically binding to the test substance, and a blocking agent, and the label is the first It is bound with one substance and blocked with the blocking agent.

In the present invention, the "label" functions as a label detected as a signal in the test line and control line. Labels according to the present invention are preferably particles, for example, metal particles or colloidal particles of gold, silver, platinum, copper, etc.; , tellurium, sulfur and other nonmetallic particles; colored latex particles including those colored with dyes; fluorescent particles such as fluorescent silica particles and europium; Gold colloidal particles are preferred because they tend to be easy to use. When the label is a particle, its size is not particularly limited. and usually 10 to 1000 nm.

In the present invention, the "first substance capable of specifically binding to the test substance (hereinafter, sometimes simply referred to as "first substance")" specifically recognizes the target test substance, Indicates a substance that can bind. As such a first substance, typically, when the test substance is an antigen, an antibody capable of specifically binding to the antigen; when the test substance is an antibody, capable of specifically binding to the antibody antigens or antibodies (antibodies against immunoglobulin molecules); when the test substance is a nucleic acid, it is a nucleic acid, an antibody, or a binding protein capable of specifically binding to the nucleic acid.

As used herein, the “antibody” may be of any isotype such as IgG, IgM, IgA, IgD, IgE, and IgY. Also, as used herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies), and antibody fragments thereof, and the antibody fragments include, for example, Fab , Fab′, F(ab′) ₂ , Fv, single-chain antibodies, heavy-chain antibodies, diabodies, and low-molecular-weight antibodies bound to antibody variable regions.

In the present invention, when the first substance is an antibody, the antibody (anti-test substance antibody) is not particularly limited as long as it has the ability to bind to the test substance, and may be a polyclonal antibody or a monoclonal antibody. or the antibody fragment. Such an anti-test substance antibody can be produced by appropriately adopting a conventionally known method or a method based thereon, and in the case of a polyclonal antibody, for example, immunization with the test substance as an immunogen It can be prepared from the serum of animals (e.g., mice, rats, hamsters, rabbits, etc.) that have been immunized, and in the case of monoclonal antibodies, for example, antibody-producing cells (animals immunized with the test substance as an immunogen spleen cells, lymph node cells, peripheral blood leukocytes, etc.) and myeloma cells fused with cells (hybridoma) to produce antibodies (representatively, methods by Kohler and Milstein (Kohler & Milstein, Nature, 256: 495, 1975)). In addition, the anti-test substance antibody can also be produced by a recombinant DNA method if the DNA encoding the antibody can be obtained. Furthermore, as the anti-test substance antibody, those generally distributed may be appropriately used.

In the present invention, the "blocking agent" covers the surface of the label to which the first substance is not bound, and the label is a sample-derived protein or other substance other than the first substance. It has a function to prevent non-specific binding to For example, when colloidal gold particles are used as the label, the surfaces of the colloidal gold particles are non-specific to the first substance (antigen, antibody, etc.), proteins derived from the sample, and multiple substances thereof. Since it has the property of being easily adsorbed, even if the test substance is not contained in the sample, the determination result may be positive (false positive). Therefore, the surface of the label to which the first substance is not bound is blocked and covered with the blocking agent. Moreover, in the present invention, the blocking agent is specifically recognized by the following third substance.

The blocking agent is not particularly limited, and those commonly used as blocking agents can be appropriately used. serum albumin (HSA), skim milk. In the present invention, antibodies, protein G, protein A, etc. can also be used as the blocking agent as long as they do not bind to either the test substance or the first substance. Among these, the blocking agent according to the present invention is preferably at least one protein (blocking protein) selected from the group consisting of casein and Escherichia coli-derived heat shock protein, and is preferably a biological protein. more preferred.

In the labeling reagent according to the present invention, the label and the first substance are bound. Physical adsorption is also included in the binding between the label and the first substance. As a method for binding the label and the first substance, a conventionally known method or a method based thereon can be appropriately adopted. (a carboxyl group, an epoxy group, a tosyl group, an amino group, a hydroxyl group, etc.) and a method of bonding through the modified group. In the binding of the label and the first substance, the compounding ratio (label: first substance) depends on the ease of binding of the first substance to the label and the first substance to be used. can be adjusted as appropriate depending on the sensitivity to the test substance, etc.

In the labeling reagent according to the present invention, the surface of the label to which the first substance is not bound is blocked with the blocking agent. As a method for blocking the surface of the label to which the first substance is not bound with the blocking agent, a conventionally known method or a method analogous thereto can be employed as appropriate. For example, the first substance (eg, anti-test substance antibody) is added to a colloidal solution) to immobilize the antibody on the surface of the label. By adding the blocking agent to this, the labeling reagent according to the present invention can be obtained by binding the blocking agent to the surface of the label to which the first substance is not bound and blocking it. In the labeling reagent according to the present invention, the compounding ratio of the first substance and the blocking agent (first substance:blocking agent) is not particularly limited. A ratio of 0.1 to 1:100 is preferred.

As a method for holding the labeling reagent in the conjugate pad according to the present invention, a sample (preferably an aqueous solution or aqueous dispersion) or a method in which the labeling reagent can be eluted when the following developing solution is developed. There is no particular limitation, and examples thereof include a method of coating or impregnating the material of the conjugate pad with a solution containing the labeling reagent, followed by drying. Examples of the solution containing the labeling reagent include a buffer solution, and examples of the buffer solution include the same buffers as those mentioned above for the pretreatment solution. The amount of the labeling reagent held in the conjugate pad according to the present invention can be appropriately adjusted depending on the form of the sample, the sensitivity of the first substance to be used to the test substance, the purpose of measurement, and the like.

Moreover, it is preferable that the conjugate pad according to the present invention further retains the non-specific reaction inhibitor in an elutable manner. The method for further retaining the non-specific reaction inhibitor is the same as described above, and when the non-specific reaction inhibitor is further retained, the type and amount thereof are appropriately adjusted depending on the type and concentration of the sample to be measured. can do.

In the immunochromatographic test strip of the present invention, when the sample contacts the conjugate pad, the labeling reagent is eluted, and when the test substance is present in the sample, the test substance and the label A complex (eg, immune complex) is formed by interaction (eg, antigen-antibody reaction) with the first substance that constitutes the reagent.

(porous membrane)
The porous membrane according to the present invention (porous membrane 120 in FIG. 1) is an insoluble carrier that functions as a chromatographic medium (stationary phase) for immunochromatography. Moreover, the porous membrane according to the present invention is also an antibody-immobilized membrane having the following test line and control line.

The porous membrane is not particularly limited as long as it is a porous membrane, and examples thereof include nitrocellulose membranes, nitrocellulose mixed ester membranes, cellulose membranes, acetylcellulose membranes, polysulfone membranes, polyethersulfone membranes, nylon membranes, and glass fibers. , nonwoven fabrics, and cloths, and among these, nitrocellulose membranes or nitrocellulose mixed ester membranes are preferred from the standpoint of ease of fabricating test lines and control lines. The speed of movement of the sample developed on such a porous membrane can be changed depending on the material, size, pore size, distribution, etc. of the porous membrane. , can be adjusted accordingly.

[Test line]
In the present invention, the test line (test line 122 in FIG. 1) possessed by the porous membrane has a second substance that can specifically bind to the test substance immobilized on the test line region of the porous membrane. and functions as the detection part of the immunochromatographic test strip of the present invention. A test line according to the present invention is arranged downstream of the conjugate pad.

In the present invention, "a second substance capable of specifically binding to a test substance (hereinafter, sometimes simply referred to as a "second substance")" specifically recognizes the target test substance, Indicates a substance that can bind. Examples of such a second substance include substances similar to those exemplified as the first substance. Further, the second substance includes the first substance constituting the test substance and the labeling reagent. It may be a substance capable of specifically binding to a complex (eg, immune complex) formed by interaction with a substance (eg, antigen-antibody reaction). The first substance and the second substance may be the same or different, but preferably do not compete in binding to the test substance depending on the recognition site of the test substance.

In the immunochromatographic test strip of the present invention, when the labeled reagent bound to the test substance to form a complex moves to the test line, the second substance immobilized on the test line passes through the test substance. Complexes are captured and the labeled reagents that make up the complexes accumulate in the test line. As a result, the test line shows coloration or the like due to the accumulation of the labeling reagent, which can be detected as a signal and the test substance in the sample can be measured.

Although the test line 122 is formed in a line shape in FIG. 1, the shape of the test line according to the present invention is not limited to this, and may be any shape such as a circle or a polygon. Among these shapes, the shape of the test line according to the present invention is preferably linear, more preferably linear with a width of 0.5 to 1.5 mm.

[Control line]
In the present invention, the control line (control line 124 in FIG. 1) possessed by the porous membrane is a control reagent immobilized on the control line region of the porous membrane, and the immunochromatographic test strip of the present invention , it functions as a control section for determining whether or not the test is successful. The control line according to the present invention is arranged downstream of the conjugate pad, and the control line is equidistant from the test line and the conjugate pad, or is located downstream of the test line. is preferably placed in

[Control reagent]
In the present invention, the control reagent immobilized on the control line is a third substance capable of specifically binding to the blocking agent. In the present invention, "a third substance that can specifically bind to a blocking agent (hereinafter, sometimes simply referred to as a "third substance")" specifically recognizes and binds to the blocking agent Indicates a substance that can Such a third substance is typically an anti-blocking agent antibody capable of specifically binding to the blocking agent. Further, it is more preferable that the third substance according to the present invention does not or hardly binds to non-specific reaction inhibitors of various biological substances.

Such a third substance is preferably an anti-blocking agent antibody, and the anti-blocking agent antibody is, for example, an anti-E. coli-derived heat shock protein when the blocking agent is an E. coli-derived heat shock protein. An antibody (anti-BPF antibody), an anti-bovine serum albumin antibody (anti-BSA antibody) when the blocking agent is bovine serum albumin, and an anti-human serum albumin antibody (anti-BSA antibody) when the blocking agent is human serum albumin anti-HSA antibody), and anti-casein antibody when the blocking agent is casein or skimmed milk. , casein, Escherichia coli-derived heat shock protein) and at least one selected from the group consisting of anti-blocking protein antibodies against these (eg, anti-casein antibody, anti-BPF antibody).

The anti-blocking agent antibody may be of any isotype such as IgG, IgM, IgA, IgD, IgE, and IgY, and may be a complete antibody or an antibody fragment. As the anti-blocking agent antibody, those derived from various animal species such as mouse, rabbit, bovine, goat, sheep, rat, alpaca, and chicken can be used.

In the immunochromatographic test strip of the present invention, when the labeling reagent (labeling reagent not captured by the test line when the control line is arranged downstream of the test line) moves to the control line, The control reagent (third substance, preferably anti-blocking agent antibody) immobilized on the control line is captured via the blocking agent, and the labeling reagent comprising the blocking agent accumulates in the control line. As a result, the control line shows coloration or the like due to the accumulation of the labeling reagent, and this can be detected as a signal to confirm normal movement of the sample on the immunochromatographic test strip. INDUSTRIAL APPLICABILITY According to the present invention, even when various biological substances are used as non-specific reaction inhibitors, it is possible to color control lines without attenuating signal intensity.

In addition, in the immunochromatographic test strip of the present invention, unlike the conventional control line detection method, it is not bound by the animal species from which the antibody constituting the labeling reagent is derived, so it can be used as a universal reagent with high versatility. can be used.

Although the control line 124 is formed in a line shape in FIG. 1, the shape of the control line according to the present invention is not limited to this, and may be circular, polygonal, or any other shape. Among these shapes, the shape of the control line according to the present invention is preferably linear, more preferably linear with a width of 0.5 to 1.5 mm.

A method for preparing the test line and the control line in the porous membrane according to the present invention, i.e., a method for immobilizing the second substance and the control reagent (third substance, preferably anti-blocking agent antibody). is not particularly limited, and a conventionally known method or a method analogous thereto can be appropriately employed, and examples thereof include a physical adsorption method and a method of covalent bonding. For example, when the porous membrane is a nitrocellulose membrane or a nitrocellulose mixed ester membrane, it is possible to physically adsorb to this, so it is preferable to adopt a physical adsorption method. The amount of the second substance to be immobilized and the control reagent can be appropriately adjusted according to their types, the type of sample to be measured, the sensitivity to the test substance or blocking agent, etc., and is usually per ¹ mm3 of the porous membrane. , each independently preferably 0.04 to 1.2 μg.

(absorbent pad)
The absorbent pad according to the present invention (absorbent pad 150 in FIG. 1) absorbs the sample and developing solution that have migrated through the porous membrane, which is a chromatography medium, and the labeled reagent and control reagent that have not been captured by the test line and control line. It is a member that has the function of absorbing. The absorbent pad according to the present invention is usually arranged downstream of the test line and the control line. The material of the absorbent pad is not particularly limited, and examples thereof include absorbent materials such as cellulose filter paper, nonwoven fabric, cloth, and cellulose acetate film.

In FIG. 1, the sample pad 140, the conjugate pad 130, the porous membrane 120, and the absorbent pad 150 are each composed of different members, but the configuration of the immunochromatographic test strip of the present invention is not limited to this. However, these members may be members that allow the sample to move by capillary action. It may also be a series of sheets of flexible material.

<Test substance measurement method using immunochromatographic test strip>
The test substance measurement method of the present invention is a method of measuring a test substance in a sample using the immunochromatographic test strip of the present invention,
contacting the sample with the conjugate pad to form a complex between the test substance in the sample and the labeling reagent;
Capturing and detecting the complex via the test substance with the second substance in the test line;
Capturing the labeling reagent with the third substance through the blocking agent in the control line;
characterized by comprising More preferably
receiving the sample on the sample pad (step 1);
contacting the sample with the conjugate pad to form a complex between the test substance in the sample and the labeling reagent (step 2);
Capturing and detecting the complex with the second substance via the test substance in the test line (step 3), and in the control line with the third substance via the blocking agent capturing the labeling reagent (step 4);
characterized by comprising

A measurement method (immunochromatographic method) using the test piece 100 of FIG. 1 will be described below as an embodiment of the test substance measurement method of the present invention.

First, a sample is dropped or applied onto the sample pad 140 to be received (step 1). The sample is as described above, and may be one prepared by adding or using the pretreatment liquid, or one to which the nonspecific reaction inhibitor is further added. In addition, the developing solution and/or the non-specific reaction inhibitor may be dropped or applied to the sample pad at the same time as or separately from the sample. Examples of the developing liquid include the same ones as those mentioned as the pretreatment liquid, although the purpose of use thereof is different from that of the pretreatment liquid. may be

The sample received by the sample pad 140 passes through the sample pad 140 and moves to the conjugate pad 130 by capillary action. Thereby, the sample is brought into contact with the conjugate pad 130 to elute the labeling reagent, and when the test substance is present in the sample, the test substance (e.g., antigen) and the labeling reagent constitute A complex (eg, immune complex) between the test substance and the labeling reagent is formed by interaction (eg, antigen-antibody reaction) with the first substance (eg, antibody against the antigen) (step 2).

Further, at this time, when the non-specific reaction inhibitor is added to the sample or conjugate pad 130, or when the non-specific reaction inhibitor is held in the conjugate pad 130 so that it can be further eluted, , when a heterophile antibody is present in the sample, the nonspecific reaction inhibitor binds to the heterophile antibody, and downstream, the heterophile antibody binds to the following first substance and / Or, it binds to a second substance to prevent a false positive determination from occurring.

The complex then moves downstream by capillary action and reaches the test line 122 (detector). Thereby, in the test line 122, the second substance (for example, antibody against the antigen) immobilized on the test line 122 captures and detects the complex via the test substance (step 3). As a result of the complex being captured by the test line 122, the test line 122 exhibits coloration or the like due to the labeling substance that constitutes the labeling reagent. A test substance in a sample can be measured.

In addition, the labeling reagent eluted from the conjugate pad (the labeling reagent not captured in the test line when the control line is arranged downstream of the test line) moves downstream due to capillary action. and reaches the control line 124 as well. Thereby, in the control line 124, the control reagent (third substance, preferably anti-blocking agent antibody) immobilized on the test line 122 captures and detects the labeling reagent via the blocking agent (step 4). In addition, in the test piece 100 of FIG. Step 3 may be performed later. As a result of the labeling reagent being captured by the control line 124, the control line 124 exhibits coloration or the like due to the labeling substance that constitutes the labeling reagent. Successful migration of the sample on the immunochromatographic strip can be determined. The criteria for determination can be appropriately set depending on the purpose of measurement, the type of test substance, and the like.

<Immunochromatography test kit>
The immunochromatographic test kit of the present invention contains at least the immunochromatographic test strip of the present invention.

In addition, the immunochromatography test kit of the present invention may further contain at least one selected from the group consisting of the pretreatment liquid, the developing liquid, and a non-specific reaction inhibitor, if necessary. Furthermore, the labeling reagent, the control reagent, the pretreatment cartridge, and the like can be combined. In addition, when an enzyme label is used as the label, a substrate, a reaction stop solution, and the like necessary for detection of the label can be included. In addition, when the competitive method is used as the detection principle, for example, a solid phase on which a solid-phased test substance or a substance that binds to the test substance is immobilized can be included. Furthermore, the immunochromatographic test kit of the present invention can further include instructions for use of the kit.

The immunochromatographic test strip, test substance measurement method, and immunochromatographic test kit of the present invention can be used not only for research but also, for example, as an in-vitro diagnostic drug, which is a basis for diagnosing a disease related to the test substance. It can be used to measure substances.

EXAMPLES The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited by the following examples.

(Example 1)
<Preparation of immunochromatographic test piece>
An immunochromatographic test piece having the structure shown in FIG. 1 was prepared according to the following (1) to (3). The prepared immunochromatography test piece has a configuration in which a porous membrane 120 made of a nitrocellulose membrane is placed on an adhesive sheet 110, and a conjugate pad 130 is placed on one end of the porous membrane 120. The conjugate pad 130 holds a labeling reagent (anti-Legionella pneumophila serotype 1 LPS rabbit polyclonal antibody-sensitized colloidal gold particles) blocked with a blocking agent (E. coli-derived heat shock protein (BPF)). In addition, an anti-Legionella pneumophila serotype 1 LPS rabbit polyclonal antibody is immobilized as an anti-test substance antibody on the test line 122 which is a detection portion located downstream of the conjugate pad 130 . Furthermore, an anti-BPF antibody is immobilized as an anti-blocking agent antibody on the control line 124 which is a control portion located downstream of the conjugate pad 130 .

(1) Preparation of test line and control line An anti-Legionella pneumophila serotype 1 LPS rabbit polyclonal antibody (anti-Legionella rabbit antibody) prepared by a conventional method was previously diluted in 20 mM phosphate buffer (pH 7.5) and added to 0.5 mg. /ml. This was applied to the test line region of a porous membrane made of nitrocellulose and dried to prepare a test line (test line 122 in FIG. 1) on which the anti-Legionella rabbit antibody was immobilized. Further, a rabbit-derived purified IgG antibody against BPF (anti-BPF rabbit antibody) prepared by a conventional method was previously diluted in 10 mM phosphate buffer (pH 7.5) to 1.5 mg/ml. This is applied to the control line region of the porous membrane and dried to prepare a control line (control line 124 in FIG. 1) on which the anti-BPF rabbit antibody is immobilized, and a porous membrane having a test line and a control line A membrane 120 was obtained.

(2) Preparation of conjugate pad First, a solution of anti-Legionella pneumophila serotype 1LPS rabbit polyclonal antibody (anti-Legionella rabbit antibody) diluted with 50 mM borate buffer (pH 9.0) is added to 1000 μL of colloidal gold solution. 100 μL was added. Next, after adding 100 μL of BPF solution as a blocking agent to this solution, the supernatant was removed by centrifugation at 10,000×g and 25° C. for 10 minutes using a centrifuge, followed by addition of 20 mM Tris-HCl ( A stock solution consisting of pH 8.2) was added. After redispersing the particles with a vortex mixer, the particles were centrifuged at 10,000×g and 25° C. for 10 minutes. Furthermore, after removing the supernatant, a storage solution consisting of 20 mM Tris-HCl (pH 8.2) was added, the particles were redispersed with a vortex mixer, and a solution of anti-Legionella rabbit antibody-sensitized colloidal gold particles (labeling reagent) was prepared. got Next, 400 μL of 20 mM Tris-HCl (pH 8.2) solution containing Normal Rabbit Globlin (NRG, non-specific reaction inhibitor) was added to 100 μL of this labeling reagent solution and mixed well. 800 μL of the resulting solution was added to a glass fiber pad, uniformly applied, and dried sufficiently to obtain a conjugate pad 130 holding the labeling reagent and NRG.

(3) Preparation of immunochromatographic test piece Place the conjugate pad 130 prepared in (2) on the porous membrane 120 having the test line and control line prepared in (1), and further sample pad 140 thereon. A glass fiber pad was placed as a In addition, an immunochromatographic test piece 100 was obtained by placing a cellulose acetate membrane as an absorbent pad 150 for absorbing the developed aqueous solution on the porous membrane 120 so as to be on the downstream side of the control line.

(Example 2)
A test line and a control line were prepared in the same manner as in Example 1 (1), except that instead of the anti-BPF rabbit antibody, a rabbit-derived purified IgG antibody against casein (anti-casein rabbit antibody) prepared by a conventional method was used. A porous membrane with A conjugate pad was prepared in the same manner as in Example 1 (2), except that a casein solution was used as the blocking agent instead of the BPF solution. Furthermore, an immunochromatographic test strip was obtained in the same manner as in Example 1 (3) except that these were used as the porous membrane 120 and the conjugate pad 130, respectively.

(Example 3)
In the same manner as in Example 1 (1), except that an anti-RSV rat monoclonal antibody (anti-RSV rat antibody) prepared by a conventional method was used instead of the anti-Legionella rabbit antibody, a porous membrane having a test line and a control line was prepared. A membrane was fabricated. A conjugate pad was prepared in the same manner as in Example 1 (2) except that the anti-RSV rat antibody was used in place of the anti-Legionella rabbit antibody and NRG was not added. Furthermore, an immunochromatographic test strip was obtained in the same manner as in Example 1 (3) except that these were used as the porous membrane 120 and the conjugate pad 130, respectively.

(Example 4)
A porous membrane having a test line and a control line was prepared in the same manner as in Example 3, except that the same anti-casein rabbit antibody as in Example 2 was used instead of the anti-BPF rabbit antibody. A conjugate pad was prepared in the same manner as in Example 3, except that a casein solution was used as the blocking agent instead of the BPF solution. Further, an immunochromatography test piece was obtained in the same manner as in Example 3 except that these were used as the porous membrane 120 and the conjugate pad 130, respectively.

(Example 5)
A porous membrane having a test line and a control line was produced in the same manner as in Example 3, except that an anti-NV mouse monoclonal antibody (anti-NV mouse antibody) prepared by a conventional method was used instead of the anti-RSV rat antibody. did. A conjugate pad was prepared in the same manner as in Example 3, except that the anti-NV mouse antibody was used instead of the anti-RSV rat antibody. Further, an immunochromatography test piece was obtained in the same manner as in Example 3 except that these were used as the porous membrane 120 and the conjugate pad 130, respectively.

(Comparative example 1)
(1) A porous membrane having a test line and a control line was prepared in the same manner as in (1) of Example 1, except that an anti-mouse IgG antibody prepared by a conventional method was used instead of the anti-BPF rabbit antibody. .

(2) First, in the same manner as in Example 1 (2), a solution of anti-Legionella rabbit antibody-sensitized colloidal gold particles (labeling reagent) was obtained. Further, mouse IgG antibody sensitization was carried out in the same manner as the solution of anti-Legionella rabbit antibody-sensitized colloidal gold particles (labeling reagent) except that mouse IgG antibody prepared by a conventional method was used instead of anti-Legionella rabbit antibody. A solution of colloidal gold particles (dummy particles) was prepared. Next, 400 μL of a 20 mM Tris-HCl (pH 8.2) solution containing NRG was added to 100 μL of the resulting labeling reagent solution and 100 μL of the dummy particle solution and mixed well. 800 μL of the resulting solution was added to a glass fiber pad, uniformly applied, and dried sufficiently to obtain a conjugate pad holding the labeling reagent, dummy particles, and NRG.

(3) Performed except that the porous membrane obtained in (1) and the conjugate pad obtained in (2) of Comparative Example 1 were used instead of the porous membrane 120 and the conjugate pad 130, respectively. An immunochromatographic test piece was obtained in the same manner as in Example 1 (3).

(Comparative example 2)
A conjugate pad was obtained in the same manner as in (2) of Comparative Example 1, except that NRG was not added. An immunochromatographic test piece was obtained in the same manner as in Comparative Example 1 except that this was used instead of the conjugate pad 130 .

(Comparative Example 3)
A porous membrane having a test line and a control line was prepared in the same manner as in Example 1 (1) except that an anti-rabbit IgG antibody prepared by a conventional method was used instead of the anti-BPF rabbit antibody. An immunochromatography test piece was obtained in the same manner as in Example 1 except that this was used instead of the porous membrane 120 .

(Comparative Example 4)
A conjugate pad was prepared in the same manner as in Comparative Example 3 except that NRG was not added, and an immunochromatography test strip was obtained in the same manner as in Comparative Example 3 except that this was used instead of the conjugate pad 130. rice field.

(Test example 1)
Signal intensity and non-specific reaction suppression effect of control line by blocking agent detection method in immunochromatography test piece using urine as sample (comparison with conventional technology)
As samples, human urine specimens (negative samples A to C) in which the absence of Legionella-derived polysaccharide antigens has been confirmed, and HARA serum was added (spiked) to each of the negative samples A to C to a concentration of 10% by mass. A total of 9 types of false-positive samples A to C and positive samples A to C obtained by adding Legionella-derived polysaccharide antigens to the negative samples A to C were used. The presence/absence of Legionella-derived polysaccharide antigens in urine specimens was confirmed using an immunochromatographic reagent Legionella kit "Immunocatch-Legionella" (manufactured by Eiken Chemical Co., Ltd.).

About 90 μL of each sample was dropped onto the sample pad of the immunochromatographic test piece prepared in Example 1 and Comparative Examples 1 to 4, and the signal of the test line was confirmed after 15 minutes. Confirmation of the signal of the test line is done visually, and the following criteria are used:
+: The coloration of the test line can be clearly confirmed (positive)
+ ↓: Slight coloration of the test line can be confirmed (weakly positive)
－ : Color development of the test line cannot be confirmed (negative)
determined by In addition, the signal of the control line was visually confirmed, and the following criteria were observed:
A: The coloration of the control line can be clearly confirmed. B: The coloration of the control line can be slightly confirmed, but it is difficult to confirm. C: The coloration of the control line cannot be confirmed. In addition, if the test line is colored in dots instead of linear, and clogging of the porous membrane is clearly confirmed, clogging will be determined as "Yes". Other than that, "none". The results obtained are shown in Table 1 below.

As shown in Table 1, in the immunochromatographic test strip of Example 1, the occurrence of positive determination (false positive) was significantly suppressed even in false-positive samples containing HARA serum but not antigens, In addition, the coloration of the control line was clearly confirmed, and it was confirmed that sufficient signal intensity could be obtained. In addition, in the immunochromatographic test strip of Example 1, the coloration of the test line and control line for the positive sample containing the antigen was clearly confirmed, and the signal intensity did not decrease significantly. It was also confirmed that the advantages of diagnostic immunoassays are not compromised.

On the other hand, in the immunochromatographic test strips of Comparative Examples 1 and 2, clogging occurred, variations in the signal intensity of the test line occurred, and false positives occurred even in negative samples. This is thought to be due to an increase in the amount of particles due to the presence of dummy particles, an increase in the background due to this, and non-specific reactions to the dummy particles. In the immunochromatographic test piece of Comparative Example 3, although there was no problem with the signal intensity of the test line, the signal intensity of the control line was significantly attenuated, making it difficult to confirm the presence or absence of sample development. It can be said that this is because NRG as a non-specific reaction inhibitor was captured in the control line and inhibited capture of the labeling reagent.

(Test example 2)
Signal intensity and non-specific reaction suppression effect of control line by blocking agent detection method in immunochromatography test piece using urine as sample (type of blocking agent)
The samples used were human urine specimens (negative samples D to F) in which the absence of Legionella-derived polysaccharide antigens had been confirmed, and HARA serum was added (spiked) to each of the negative samples D to F to a concentration of 10% by mass. A total of 9 types of false-positive samples D to F and positive samples D to F obtained by adding Legionella-derived polysaccharide antigens to the negative samples D to F were used. The presence/absence of Legionella-derived polysaccharide antigens in urine specimens was confirmed using an immunochromatographic reagent Legionella kit "Immunocatch-Legionella" (manufactured by Eiken Chemical Co., Ltd.).

About 90 μL of each sample was dropped onto the sample pad of the immunochromatographic test piece prepared in Examples 1 and 2, and after 15 minutes, the signals of the test line and the control line were confirmed and judged according to the criteria described in Test Example 1. . The results obtained are shown in Table 2 below.

As shown in Table 2, in the immunochromatographic test strips of Examples 1 and 2, the occurrence of false positives was significantly suppressed even in false positive samples containing HARA serum but not containing antigens, and It was confirmed that the signal intensity of the control line could be made sufficiently strong. In addition, in any of the immunochromatographic test strips of Examples 1 and 2, the signal intensity of the test line and control line for the positive sample containing the antigen did not decrease significantly, which contributes to rapid examination and diagnosis. It was also confirmed that the advantages of the immunoassay method are not compromised, and it was confirmed that the blocking agent in the present invention is not limited to BPF.

(Test example 3)
Signal intensity and non-specific reaction suppression effect of control line by blocking agent detection method in immunochromatographic test piece using nasal swab as sample Negative samples G to I suspended in an extraction solution (HEPES buffer) containing a rat antibody (prepared by a conventional method) as a reaction inhibitor, and 10% by mass of human anti-rat antibody serum in each of the negative samples G to I. False-positive samples G to I were added (spiked) in such a manner as to obtain positive samples G to I, and positive samples G to I were added to the negative samples G to I with RSV antigen, respectively. The presence/absence of the RSV antigen in the sample was confirmed using the PCR reagent "Respiratory Viral Panel" (manufactured by GenMark).

About 90 μL of each sample was dropped onto the sample pad of the immunochromatography test piece prepared in Examples 3 and 4, and after 15 minutes, the signals of the test line and control line were confirmed and judged according to the criteria described in Test Example 1. . The results obtained are shown in Table 3 below.

As shown in Table 3, in the immunochromatographic test strips of Examples 3 and 4, even false positive samples containing human anti-rat antibody serum but not containing antigens significantly suppressed the occurrence of false positives. Moreover, it was confirmed that the signal intensity of the control line can be sufficiently increased. In addition, in any of the immunochromatographic test strips of Examples 3 and 4, the signal intensity of the test line and control line for the positive sample containing the antigen did not decrease significantly, which contributes to rapid examination and diagnosis. It was also confirmed that the advantages of the immunoassay method are not compromised, and it was confirmed that the present invention is also effective when nasal swab samples are used as samples.

(Test example 4)
Signal strength and non-specific reaction inhibition effect of control line by blocking agent detection method in immunochromatographic test piece using stool sample As a sample, human stool specimen confirmed the absence of NV antigen was used as a non-specific reaction inhibitor. HAMA serum was added (spike ), and positive samples J to L in which NV antigen was added to the negative samples J to L, respectively. The presence/absence of NV antigen was confirmed using an immunochromatographic reagent, "Immunocatch-Noro" (manufactured by Eiken Chemical Co., Ltd.).

About 90 μL of each sample was dropped on the sample pad of the immunochromatography test piece prepared in Example 5, and after 15 minutes, the signals of the test line and control line were confirmed and judged according to the criteria described in Test Example 1. The results obtained are shown in Table 4 below.

As shown in Table 4, in the immunochromatographic test strip of Example 5, even in the false-positive sample containing HAMA serum but not containing the antigen, the occurrence of false-positive was significantly suppressed, and the control line It was confirmed that the signal intensity of the signal can be sufficiently strong. In addition, in the immunochromatographic test strip of Example 5, the signal intensity of the test line and the control line for the positive sample containing the antigen did not decrease significantly. It was also confirmed that the advantages of the method are not impaired, and it was confirmed that the present invention is effective even when a stool sample is used as a sample.

According to the present invention, in the immunochromatographic method, there is no need to separately prepare reagents other than the labeling reagent and the control reagent in order to detect the signal in the control line, and non-specific reaction suppression against heterophile antibodies in the sample It is possible to provide an immunochromatographic test strip with a control line signal formation mechanism that does not affect the signal intensity in the control line even in the presence of an agent, and a method for measuring a test substance and an immunochromatographic test kit using the same. becomes.

DESCRIPTION OF SYMBOLS 100... Test piece, 110... Sheet, 120... Porous membrane, 130... Conjugate pad, 140... Sample pad, 150... Absorption pad, 122... Test line, 124... Control line

Claims (10)

An immunochromatographic test strip for measuring a test substance in a sample,
Equipped with conjugate pads, test lines, and control lines,
The conjugate pad retains an elutable labeling reagent containing a first substance capable of specifically binding to the test substance, a blocking agent, and a label,
the label is bound to the first substance and blocked with the blocking agent;
A second substance that can specifically bind to the test substance is immobilized on the test line,
A third substance that can specifically bind to the blocking agent is immobilized on the control line,
An immunochromatographic test strip characterized by: The immunochromatography according to claim 1, wherein the blocking agent is at least one protein selected from the group consisting of casein, E. coli-derived heat shock protein, bovine serum albumin, human serum albumin, and skim milk. graphic specimen. The sample contains a non-specific reaction inhibitor, or the conjugate pad is characterized in that the non-specific reaction inhibitor is held so that it can be further eluted, immunochromatography according to claim 1 or 2 graphic specimen. The non-specific reaction inhibitor is at least one selected from the group consisting of normal mouse globulin, normal rabbit globulin, normal bovine globulin, normal goat globulin, normal sheep globulin, normal rat globulin, normal alpaca globulin, and normal chicken globulin. The immunochromatographic test strip according to claim 3, characterized in that wherein said third substance is at least one organism-derived anti-blocking agent antibody selected from the group consisting of mouse, rabbit, cow, goat, sheep, rat, alpaca, and chicken; The immunochromatographic test strip according to any one of Items 1 to 4. A method for measuring a test substance in a sample using the immunochromatographic test strip according to any one of claims 1 to 5,
contacting the sample with the conjugate pad to form a complex between the test substance in the sample and the labeling reagent;
Capturing and detecting the complex via the test substance with the second substance in the test line;
Capturing the labeling reagent with the third substance through the blocking agent in the control line;
A method for measuring a test substance, comprising: Further comprising the step of adding a non-specific reaction inhibitor to the sample or the conjugate pad, or the non-specific reaction inhibitor is held in the conjugate pad so that it can be further eluted, The method for measuring a test substance according to claim 6. An immunochromatographic test kit comprising the immunochromatographic test strip according to any one of claims 1 to 5. The immunochromatographic test kit according to claim 8, further comprising a non-specific reaction inhibitor. The non-specific reaction inhibitor is at least one selected from the group consisting of normal mouse globulin, normal rabbit globulin, normal bovine globulin, normal goat globulin, normal sheep globulin, normal rat globulin, normal alpaca globulin, and normal chicken globulin. The immunochromatography test kit according to claim 9, characterized in that it is

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