阅读说明：本技术 用于检测含红细胞的样品中的对象物的免疫色谱用测试条、及使用该测试条的免疫色谱 (Test strip for immunochromatography for detecting object in sample containing red blood cells, and immunochromatography using same ) 是由 河野景吾 吉田万友美 山本光章 横川伸也 浅见麻里惠 于 2014-12-16 设计创作，主要内容包括：本发明的课题在于,提供一种使用聚凝胺作为红细胞凝集剂、使用胶体金缀合物作为检测试剂时,将全血中的红细胞凝集并分离除去并且不引起胶体金的凝集的免疫色谱用测试条；以及利用免疫色谱的检测方法。本发明人们为了解决上述课题,并非进行聚阴离子的种类、量的选定,而是从完全不同的观点出发,审视了此前的试剂构成本身并对各要素进行了深入研究,结果惊奇地发现,通过使用特定的添加剂,在不基于聚阴离子进行中和的情况下也能够抑制胶体金的凝集。(The subject of the present invention is to provide a test strip for immunochromatography, which agglutinates and separates erythrocytes in whole blood and removes them without causing agglutination of colloidal gold, when polybrene is used as an erythrocyte agglutinating agent and a colloidal gold conjugate is used as a detection reagent; and a detection method using immunochromatography. In order to solve the above problems, the present inventors have made extensive studies on the respective elements by examining the conventional reagent constitution itself from a completely different viewpoint, not selecting the kind and amount of polyanion, and as a result, have surprisingly found that the aggregation of colloidal gold can be suppressed without neutralizing with polyanion by using a specific additive.)

1. A detection method using immunochromatography, comprising the steps of:

(i) a step of supplying a sample to a sample supply portion of the test strip,

the test strip has a film composed of a porous body provided with at least a sample supply portion, an expansion portion, and a detection portion,

a conjugate holding portion which holds a conjugate containing colloidal gold to which an antibody against the object is fixed so as to be capable of elution, in a part of the developed portion, and a detection portion to which an immobilized antibody is fixed, in a part of the developed portion and on a downstream side of the conjugate holding portion;

(ii) bringing polybrene into contact with the sample upstream of the sample supply portion to aggregate blood-derived components in the sample;

(iii) (iii) separating and removing the aggregate obtained in the step (ii) from the sample;

(iv) (iv) a step of bringing the sample component separated and removed of the agglutinate obtained in the step (iii) into contact with a colloidal gold-containing conjugate in the presence of an additive having an ability to inhibit aggregation of the conjugate by the polybrene, wherein the additive is 1 or 2 or more compounds selected from the group consisting of (a) a salt of a metal having a valence of 2, (B) a salt of sulfuric acid and a metal or onium, (C) a salt of sulfurous acid and a metal, and (E) a compound of a poly-basic amino acid;

(v) (iii) detecting the complex of the target substance and the conjugate in the sample component obtained in the step (iv) in the detection section.

2. The detection method according to claim 1, wherein the additive is contained in a conjugate holding portion.

3. The detection method according to claim 1 or 2, wherein a polyanion for neutralizing a cation of polybrene as a red blood cell agglutinating agent is not contained as a neutralizing agent.

4. The detection method according to claim 1 or 2, wherein the sample is a heparin-containing specimen or an EDTA-containing specimen.

5. The method according to claim 3, wherein the sample is a heparin-containing specimen or an EDTA-containing specimen.

Technical Field

The present invention relates to a test strip for immunochromatography for detecting an object in a sample containing red blood cells, and an immunochromatography using the same. More specifically, the present invention relates to: a test strip treated with a specific additive so that a colloidal gold conjugate as a detection reagent is not aggregated non-specifically due to the presence of a red blood cell agglutinating agent containing a polycation as an active ingredient even when the red blood cell agglutinating agent coexists in a measurement system; and immunochromatography using the test strip.

Background

In recent years, in clinics and small-scale hospitals, there is an increasing demand for "trying to obtain examination results during patient examination", and there is a shift from the examination performed outside the former entrusted hospital to Point of Care Testing (POCT). With the spread of POCT, a device containing a lateral flow immunochromatographic test strip has been used as an in vitro diagnostic reagent. The immunochromatographic test strip is capable of detecting an object in a sample by a simple operation of directly dropping a test sample (hereinafter, sometimes referred to as "sample") such as blood or urine onto the test strip without preparing a reagent at the time of examination, and is very useful for analyzing the object to be detected simply and quickly.

The immunochromatographic test strip (hereinafter, sometimes referred to as "test strip") is generally a porous membrane provided with a sample supply part, a development part, and a detection part, and has the following structure: a detection reagent such as a labeled antibody (hereinafter, sometimes referred to as "conjugate") against a detection target is held at the development initiation site of the development part so as to be eluted and developed, and after contacting a sample, the detection reagent can reach the detection part through the development part, and further, an immobilized antibody is immobilized on a part of the development part, thereby constituting the detection part. Here, when the sample is dropped into the sample supply portion, the detection target substance in the sample specifically binds to the labeled antibody to form a complex, and the complex gradually develops in the downstream direction in the development portion and further binds to the immobilized antibody. Therefore, by detecting a sandwich-type complex formed of the labeled antibody, the detection target substance, and the immobilized antibody in the antibody-immobilized portion, the detection target substance can be qualitatively or quantitatively analyzed. An example of the labeling substance constituting the conjugate is a colloidal gold particle, and qualitative detection can be performed by a color reaction of the colloidal gold particle. Further, the detection target substance in the sample can be quantitatively detected based on the degree of color development.

However, when the sample is whole blood, there are problems as follows: red blood cells in whole blood cannot migrate through the porous membrane to block the membrane pores, preventing the sample from developing. Therefore, when whole blood is used as a sample, red blood cells need to be separated and removed from the whole blood in advance, and as such a method, the following methods are known: a method in which erythrocytes are sedimented and removed by centrifugation before measurement; a method in which erythrocytes are agglutinated by a erythrocyte separating agent and removed by filtration before or at the start of measurement.

Examples of the erythrocyte separating agent include hydrabamine (which is distributed under the trade name polybrene and hereinafter may be abbreviated as polybrene) (patent documents 1, 2, and 3).

Patent document 1 discloses polybrene as an example of a red blood cell agglutinating agent made of a synthetic water-soluble polymer.

Patent document 2 describes a chromatographic blood cell separation membrane made of glass fiber, which contains polybrene as a substance capable of agglutinating red blood cells. And discloses the following techniques: in the case of the polybrene alone, hemolysis is accompanied when blood passes through the blood cell separation membrane, and in order to avoid the hemolysis, the blood cell separation membrane is coated with PVA.

As described above, polybrene is widely known as a red blood cell agglutinating agent, but when polybrene is used for immunochromatography using a metal conjugate as a detection reagent, there is a problem that not only red blood cells in whole blood are agglutinated but also the metal conjugate is agglutinated (patent document 3).

Patent document 3 discloses a technique for preventing aggregation of such a metal conjugate. That is, an immunochromatography analysis apparatus has been developed in which polycations such as polybrene as a red blood cell separating agent are bound to the upstream side of a chromatographic carrier, and polyanions for neutralizing the polycations are bound to the downstream side thereof. According to this technique, the positive charge of the polycation is neutralized by the negative charge of the polyanion, and thus the selenium-containing metal conjugate can be prevented from agglutinating.

Prior art documents

Patent document

Patent document 1: japanese laid-open patent publication No. 3-205563

Patent document 2: japanese laid-open patent publication No. 5-099918

Patent document 3: japanese Kohyo publication No. 2002-509254

Disclosure of Invention

Problems to be solved by the invention

When immunochromatography is performed using whole blood as a sample, polybrene as a red blood cell agglutinating agent, and a colloidal gold conjugate as a detection reagent, the present inventors have attempted to neutralize the positive charge of polybrene by adding a polyanion for preventing agglutination of the colloidal gold conjugate as disclosed in patent document 3. However, although agglutination and separation removal of erythrocytes in whole blood are achieved, agglutination of colloidal gold conjugates cannot be prevented. The reason for this is not clear, but may be due to the difference between the selenium conjugate and the colloidal gold conjugate in patent document 3, the kind of polyanion, and the like.

The subject of the present invention is to provide a test strip for immunochromatography, which agglutinates and separates and removes erythrocytes in blood and does not cause agglutination of a colloidal gold conjugate when polybrene is used as an erythrocyte agglutinant containing a polycation as an active ingredient and a colloidal gold conjugate is used as a detection reagent; and providing an immunochromatography using the test strip.

Means for solving the problems

The present inventors have conducted extensive studies on the respective constituent elements while examining the reagent constitution itself from a completely different viewpoint without selecting the species or amount of the polycation which is a substance causing the problem in order to solve the above-mentioned problems, and as a result, have surprisingly found that there is an additive having the ability to inhibit the aggregation of a colloidal gold conjugate by a polybrene, and found that the aggregation of a colloidal gold conjugate can be inhibited without neutralizing the polycation with a polyanion by using the additive, and thus completed the present invention. That is, the present invention has the following configuration.

[1] A test strip for immunochromatography having the following constitution.

(1) Has a membrane composed of a porous body having at least a sample supply section, an expansion section, and a detection section,

a conjugate holding portion which holds an anti-detection-target-object antibody conjugate labeled with colloidal gold so as to be eluted is provided in a part of the developed portion, and a detection portion to which an immobilized antibody is fixed is provided in a part of the developed portion and on a downstream side of the conjugate holding portion;

(2) at least a part of the upstream side of the conjugate holding section from the sample supply section to the development section contains an additive for inhibiting agglutination of the conjugate by polybrene as a red blood cell agglutinant, in a manner capable of contacting with polybrene supplied through the sample supply section.

[2] The test strip according to [1], wherein polybrene is contained in the sample supply portion.

[3] The test strip according to [1] or [2], wherein the aforementioned additive is contained in the conjugate holding portion.

[4] The test strip according to any one of [1] to [3], wherein a polyanion as a neutralizing agent for neutralizing a cation of polybrene as a red blood cell agglutinating agent is not contained.

[5] The test strip according to any one of [1] to [4], wherein the additive is 1 or 2 or more compounds selected from the group consisting of compounds belonging to (A) a salt of a 2-valent metal, (B) a salt of sulfuric acid and a metal or onium, (C) a salt of sulfurous acid and a metal, (D) a carboxylic acid chelate compound, and (E) a poly basic amino acid.

[6] A detection method using immunochromatography, comprising the steps of:

(A) a step of supplying a sample to a sample supply portion of the test strip,

the test strip has a film composed of a porous body provided with at least a sample supply portion, an expansion portion, and a detection portion,

a conjugate holding portion which holds a conjugate containing colloidal gold to which an antibody against the object is fixed so as to be capable of elution, in a part of the developed portion, and a detection portion to which an immobilized antibody is fixed, in a part of the developed portion and on a downstream side of the conjugate holding portion;

(B) bringing the polybrene into contact with the sample at or upstream of the sample supply portion to aggregate blood-derived components in the sample;

(C) separating and removing the aggregate obtained in the step (B) from the sample;

(D) a step of bringing the sample component separated and removed of the agglutinate obtained in the step (C) into contact with a colloidal gold-containing conjugate in the presence of an additive having an ability to inhibit aggregation of the conjugate by polybrene;

(E) and (D) detecting the complex of the target substance and the conjugate in the sample component, which is obtained in the step (D), in the detection section.

[7] The detection method according to [6], wherein the polybrene is contained in the sample supply part.

[8] The detection method according to [6] or [7], wherein the additive is contained in the conjugate holding portion.

[9] The detection method according to any one of [6] to [8], wherein a polyanion for neutralizing a cation of polybrene as a red blood cell agglutinant is not contained.

[10] The detection method according to any one of [6] to [9], wherein the sample is a heparin blood collection subject or an EDTA blood collection subject.

[11] The detection method according to any one of [6] to [10], wherein the additive is 1 or 2 or more compounds selected from the group consisting of (A) a salt of a 2-valent metal, (B) a salt of sulfuric acid and a metal or onium, (C) a salt of sulfurous acid and a metal, (D) a carboxylic acid chelate, and (E) a compound of a poly-basic amino acid.

Effects of the invention

According to the present invention, in immunochromatography using a polybrene as a red blood cell agglutinating agent and a colloidal gold conjugate as a detection reagent, by using an additive having an ability to inhibit agglutination of the colloidal gold conjugate by the polybrene, only red blood cells can be agglutinated and separated and removed from a sample without agglutinating the colloidal gold conjugate, and the colloidal gold conjugate can exert its original function as the detection reagent, and therefore, a detection target in the sample can be accurately detected and measured. Further, since the addition of a polyanion as a polycation neutralizer is not an essential condition, the reagent constitution can be extremely simplified, and there are also advantages in that: the factors that bring unexpected effects to the immune reaction, the spreading of the test object on the test strip, and the like are reduced.

Drawings

Fig. 1 is a graph showing the measurement results of the maximum absorption wavelength of a mixed solution of a polybrene solution and a conjugate solution to which an additive candidate substance of the present invention is added (example 1).

FIG. 2 is a schematic structural view of the test strip for immunochromatography of the present invention.

FIG. 3 is a graph showing a comparison between immunochromatography and the LTIA method using the additive of the present invention (example 2).

FIG. 4 is a graph showing the comparison of the detection sensitivity between the additive formulation of the present invention and a conventional formulation (example 3).

Detailed Description

(test strip for immunochromatography)

The immunochromatographic test strip of the present invention is a porous membrane provided with at least a "sample supply section", a "development section", and a "detection section", and has the following structure: the labeled antibody against the detection target is held in the development start site of the development part so as to be eluted, and can reach the detection part through the development part after contacting the sample, and the immobilized antibody is immobilized on a part of the development part to constitute the detection part.

As an example of embodying these, there can be cited a test strip including: a sample pad serving as a sample supply part; a conjugate pad that holds a labeled antibody against a detection target in an elutable manner and serves as a part of a development section; a porous membrane having immobilized antibodies locally immobilized thereon and serving as a development part and a detection part. That is, a typical immunochromatographic test strip of the present invention has the following configuration.

(1) Sample pad to which sample is supplied

(2) A conjugate pad disposed downstream of the sample pad and holding a conjugate in an elutable manner, the conjugate having a colloidal gold surface sensitized with a first antibody

(3) A porous membrane disposed downstream of the conjugate pad and having a second antibody immobilized thereon, the second antibody being bound to a complex of the conjugate and the detection target

Here, the sample pad, the conjugate pad, and the porous membrane may be constituted as the carriers, or 2 carriers may be constituted as 1 carrier, and any of these may be included as long as the sample pad, the conjugate pad, and the porous membrane are constituted in this order from upstream to downstream.

In addition to the above configuration, the immunochromatographic test strip includes a configuration in which at least one of an absorbent pad and a 3rd pad is further mounted. The test strip is typically arranged on a solid support such as an adhesive sheet made of plastic. The solid phase support is constituted by a substance that does not inhibit the capillary flow of the sample, and it is needless to say that the component of the adhesive is also a substance that does not inhibit the capillary flow of the sample. In addition, a process of laminating a polyester film or the like on a test strip may be performed for the purpose of improving the mechanical strength of the porous membrane to which the antibody is fixed and preventing evaporation (drying) of water in the analysis.

(erythrocyte agglutination agent)

The erythrocyte agglutination agent used in the present invention may be a known polycationic erythrocyte agglutination agent. Among them, polybrene is preferable. Polybrene, also known under the chemical name hydrabamine, is 1 cationic polymer assigned CAS number 28728-55-4.

In the present invention, polybrene is used for aggregating erythrocytes in whole blood as a sample. As a method of using polybrene, polybrene may be added to a diluted solution obtained by diluting a sample or may be added directly to the sample, and a sample supply part (sample pad) of an immunochromatographic strip may contain polybrene in advance. In such a manner of use, polybrene contacts whole blood to agglutinate red blood cells in the whole blood. In an immunochromatographic strip, when a sample passes through a filter constituting a sample supply part, most of the erythrocyte aggregates remain on the sample supply part and are removed. In the present invention, in order to remove the erythrocyte aggregates in the sample supply section and to more reliably reduce the supply to the development section, it is desirable to use a 3rd pad (blood cell separation membrane) in combination as described below.

The amount of polybrene added may be an amount that can agglutinate and separate and remove erythrocytes in whole blood so that a detection target substance in a whole blood sample can be developed as desired, and for example, when it is contained in a sample pad in advance, it is preferably 0.25% or more, more preferably 0.25 to 2% with respect to the amount of sample liquid at the time of dropping the sample. From the viewpoint of producing the sample pad, the concentration in the solution permeated into the pad is preferably 0.5% or more, and more preferably 0.5 to 4%.

In the present invention, the polyanion as a neutralizing agent for neutralizing the cation of polybrene as an erythrocyte agglutinant, which is described in patent document 3, is not included, but the presence of the polyanion generally used is not excluded as necessary within a range not affecting the reaction system without departing from the object of the present invention.

(additives)

In the present invention, a red blood cell agglutinating agent (polybrene) is used for the purpose of agglutinating red blood cells in a whole blood sample, but the polybrene has a problem of agglutinating not only red blood cells but also a colloidal gold conjugate. In the present invention, an additive is required to avoid such aggregation of the colloidal gold conjugate.

The additive used in the present invention may be any additive as long as it has the ability to inhibit the agglutination of the colloidal gold conjugate by the polybrene.

For example, an additive having such ability is added to a solution containing a colloidal gold conjugate and polybrene, and the agglutination inhibition reducing effect of the conjugate is observed, whereby the additive of the present invention can be selected. More specifically, the selection method is as follows: an additive solution as a selected object is added in advance to the colloidal gold conjugate, a polybrene solution is added thereto, and the maximum absorption wavelength is measured, and a substance having a smaller maximum absorption wavelength than that in the case of no additive is selected.

In particular, it is surprising that only one of the two polyanions, colloidal gold conjugate and red blood cell, can be selectively aggregated to inhibit the aggregation of the other polyanion in the coexistence state.

The additive selected by the above-mentioned selection method includes 1 or 2 or more compounds selected from the group consisting of (a) a salt of a metal having a valence of 2, (B) a salt of sulfuric acid with a metal or onium, (C) a salt of sulfurous acid with a metal, (D) a carboxylic acid chelate, and (E) a compound of a polybasic amino acid.

(A) Salts of 2-valent metals

Salts of 2-valent metals can further be classified as: (A1) salts of a metal having a valence of 2 with an acid, (A2) salts of a metal having a valence of 2 with a halogen element. Here, as the metal having a valence of 2, magnesium, calcium, nickel, zinc, and the like can be exemplified. Examples of the acid include inorganic acids such as sulfuric acid and nitric acid, and organic acids such as acetic acid, and examples of the halogen element include chlorine and bromine. Of these, magnesium sulfate, magnesium nitrate and magnesium acetate are suitable as specific compounds of (a1), and magnesium chloride, calcium chloride, zinc chloride, manganese chloride and nickel chloride are suitable as specific compounds of (a 2).

(B) Salts of sulfuric acid with metals or onium

Examples of the salt of (B1) sulfuric acid with a metal include: salts of sulfuric acid with a metal having a valence of 1 or salts of sulfuric acid with a metal having a valence of 2, among which potassium sulfate and magnesium sulfate (repeated with A1) are preferable. The salt of (B2) sulfuric acid and onium is preferably ammonium sulfate.

(C) Salts of sulfurous acid with metals

As the salt of sulfurous acid with a metal, sodium sulfite is preferred.

(D) Carboxylic acid chelate compounds and salts thereof

The carboxylic acid chelates can then be classified as: (D1) dicarboxylic acid chelate compounds, (D2) tricarboxylic acid chelate compounds, (D3) tetracarboxylic acid chelate compounds, (D4) pentacarboxylic acid chelate compounds. As the (D1) dicarboxylic acid chelate compound, oxalic acid, succinic acid, tartaric acid are preferable, as the (D2) tricarboxylic acid chelate compound, citric acid is preferable, as the (D3) tetracarboxylic acid chelate compound, ethylenediaminetetraacetic acid (EDTA) is preferable, and as the (D4) pentacarboxylic acid chelate compound, diethyltriaminepentaacetic acid (DTPA) is preferable. These chelates may be used in the form of the free acid or the metal salt. Specific examples of the metal salt form include: trisodium citrate, EDTA-2Na, and the like. In addition to the above, a compound obtained by modifying or changing a part of the structure of the above compound, such as glycol ether diamine tetraacetic acid (EGTA), may be used.

(E) Poly basic amino acid

Examples of the poly-basic amino acid include: Poly-L-arginine is preferably used in the form of poly-L-arginine hydrochloride. In patent document 3, it is completely unexpected that poly-L-arginine hydrochloride is a compound classified as an erythrocyte separating agent having a positive charge, similarly to polybrene, and when combined with polybrene, not only does it not enhance the effect of agglutination of the colloidal gold conjugate by polybrene, but it reduces the effect.

The additive may be added in an amount that can suppress aggregation of the colloidal gold conjugate due to the polybrene, and for example, the additive may be added in an amount of 1 to 100mmol/L, preferably 2 to 50mmol/L, 10 to 50mmol/L, based on the concentration in the solution that has permeated into the sample pad and/or the conjugate pad. The type, amount and concentration of the additive can be appropriately set according to the target substance, the desired sensitivity, and the like.

The specific additive of the present invention may be used in any form as long as it can inhibit the agglutination of the colloidal gold conjugate by the polybrene as the erythrocyte agglutinant, and the test strip for immunochromatography may be impregnated at least partially in the conjugate fixing site from the sample supply portion to the development portion so as to be able to contact with the polybrene component. Therefore, the sample supply portion may be the only portion, or the entire portion from the sample supply portion to the development portion may be the only portion.

The method of including the additive in the sample supply and/or conjugate pad includes a method of including a liquid additive in the pad, and a method of immersing the additive in the pad and then drying the same, thereby allowing the additive to adhere to the pad in a dried state.

In addition, the specific additive of the present invention may be added to a sample diluent and used.

In the present invention, as described above, it is necessary to use the additive in a specific manner of use, but this does not prevent the use of other additives than the above for other purposes or the use of other manners than the above. For example, it is needless to say that the form of impregnating the conjugate immobilized site from the sample supply part to the development part with the specific additive of the present invention and impregnating the antibody immobilized site of the development part with additives other than those described above is also included in the scope of the present invention.

(colloidal gold)

The colloidal gold used as the label in the present invention may be any colloidal gold that can sensitize (immobilize) an antibody to form a conjugate and can function as a label in a method of detecting an object (antigen) in a sample by bringing the conjugate into contact with the sample.

As the colloidal gold, colloidal platinum is also included in the colloidal gold of the present invention in addition to colloidal gold.

It is known that the particle size of the colloidal gold particles greatly affects the detection sensitivity, and for example, when the particles are used while being held in a test strip for immunochromatography, the particle size of the colloidal gold particles is preferably 20 to 60nm, more preferably 30 to 50nm, and particularly preferably 40 nm. The colloidal gold can be produced by a known method, for example, by adding dropwise an aqueous solution of trisodium citrate or an aqueous solution of triammonium citrate to a heated aqueous solution of tetrachlorogold (III) and stirring. In the present specification, colloidal gold may be referred to as colloidal gold particles, but both have the same meaning.

(sensitization of antibody to colloidal gold)

The immobilization of an antibody against a detection target to colloidal gold is generally performed by physical adsorption. In this case, the concentration of the antibody is preferably adjusted to a concentration of 1 to 5. mu.g/mL of the buffer. The kind and pH of the buffer is preferably 2mmol/L phosphate buffer (pH 6.5-8) or 2-10 mmol/L TrisTris-HCl buffer (pH 7-9), more preferably 2-10 mmol/L LTris-HCl buffer (pH 7-7.5), but the use of other buffers is also included, and is not limited thereto. In the present specification, colloidal gold having an antibody against a detection target and a control antibody (or antigen) immobilized thereon as described above is referred to as a "conjugate".

(closed)

For the conjugates of the present invention, the regions of the colloidal gold surface to which no antibody is bound may also be blocked with a blocking agent.

As the blocking agent of the colloidal gold conjugate, a component derived from a living organism is generally used, and as the component derived from a living organism, any component may be used as long as it is derived from a living organism and has a blocking effect, and for example, an animal protein and a peptide derived from an animal protein are exemplified. Specifically, there may be mentioned: BSA (bovine serum Albumin), microbial Block peptide fragment (TOYOBO), NEO PROTEIN SAVER (TOYOBO) from fibroin (sericin hydrolysate), StartingBlock ^TM(PBS) Blocking Buffer (manufactured by PIERCE) and Stabil Coat ^TM(manufactured by SurModics Inc.) and Casein.

The concentration of the component derived from a living organism may be determined as appropriate depending on the component used. For example, after adding an antibody solution to a colloidal gold solution adjusted to 1OD/mL and mixing, the biological component is added to the mixed solution in a final concentration range of 0.1 to 10%, and the mixture is blocked, more preferably in a range of 0.2 to 5%.

Further, it is also possible to use a mixture of a non-biological component and a biological component as a blocking agent for colloidal gold.

(detection reagent)

In the present invention, "detection reagent" specifically means a solution containing at least a conjugate.

The detection reagent may contain, for example, 1 or more kinds of a stabilizing agent, a dissolution assistant, and the like for the purpose of maintaining the conjugate in a stable state to promote a specific reaction between the antibody immobilized on the conjugate and the detection target substance when mixed with the sample, or to dissolve and flow the conjugate rapidly and efficiently. Examples of the stabilizer and dissolution aid include: bovine Serum Albumin (BSA), sucrose, casein, amino acids, and the like.

In addition, for the purpose of improving the detection sensitivity, a known sensitizer such as 2-methacryloyloxyethylphosphocholine may be contained in the detection reagent, if necessary.

Further, the detection reagent may contain Ca as necessary ²⁺EDTA, EGTA and the like as chelating agents for ions.

In the present specification, the terms "detection" and "measurement" include the presence of a detection target and/or the quantification of a detection target, and are to be interpreted in the broadest sense, and are not to be interpreted in a limiting sense.

(Diluent)

In the present invention, when the sample needs to be diluted depending on the concentration of the detection target in the sample, a diluent may be used. The diluent may have any composition as long as it does not significantly inhibit the antigen-antibody reaction, does not significantly promote the reaction to cause the label to excessively aggregate, and thus does not develop poorly in the capillary phenomenon, or does not make the signal of the antigen-antibody reaction corresponding to the antigen concentration undetectable.

Examples of the diluent having such an action include: purifying the water; physiological saline; a low concentration buffer solution having a pH of 6.0 to 10.0, for example, 10 to 20mmol/L phosphate buffer solution, 10 to 20mmol/L Tris-hydrochloric acid buffer solution, and 10 to 20mmol/L Bis-Tris buffer solution. In addition, surfactants may also be added to these dilutions for the purpose of controlling the speed of sample development on the test strip.

As described above, the diluent of the present invention may contain polybrene as an erythrocyte agglutinant in advance.

As described above, the diluent of the present invention may further contain an additive having an agglutination-inhibiting ability, which is an ability to inhibit agglutination of the colloidal gold conjugate by the polybrene.

(sample pad)

In the present invention, the "sample pad" is a portion serving as a sample supply portion for receiving a sample, and may include any pad as long as it is a substance or form capable of absorbing a liquid sample in a state of being formed into a pad and allowing components of the liquid and an object to be detected to pass therethrough.

As described above, the sample pad of the present invention may contain a red blood cell agglutinating agent in advance. In this case, at least a part of the sample pad may be contained, and the entire sample pad may be contained.

As described above, the sample pad of the present invention may contain a specific additive in advance. In this case, at least a part of the sample pad may be contained, and the entire sample pad may be contained. Here, when the sample pad of the present invention contains both the erythrocyte agglutination agent and the specific additive in advance, both may be contained in the same portion of the sample pad, or may be contained in different portions so as not to coexist. In addition, both of them may be included in the entire sample pad.

Specific examples of materials suitable for the sample pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dried paper, pulp, fabric, and the like. Glass fiber mats are suitable. The sample pad may also be provided with the function of a conjugate pad described later. The sample pad may contain a commonly used blocking reagent if necessary within a range that does not affect the reaction system without departing from the object of the present invention.

(conjugate pad)

In the present invention, "conjugate pad" means: a detection reagent that specifically reacts with the detection target is impregnated in a material suitable for a conjugate pad described later and dried. The conjugate pad has the following functions: when the sample passes through the conjugate pad, the detection reagent and the detection object form a complex. The conjugate pad may be configured in such a manner that it is itself in contact with the antibody-immobilized membrane. Or may be configured in the following manner: is disposed in contact with the aforementioned sample Pad, receives the sample that has passed through the sample Pad based on capillary flow, and then transfers the sample to another Pad (hereinafter sometimes referred to as "3 rd Pad") that is in contact with a face of the conjugate Pad that is different from the contact face with the aforementioned sample Pad based on capillary flow. The selection of one or more sites of the sample pad and the conjugate pad, and the manner in which the selected sites are disposed on the antibody-immobilized membrane can be appropriately changed.

In addition, as described above, the conjugate pad of the present invention may further contain a specific additive in advance. In this case, the conjugate pad may be included in a part of the conjugate pad including at least the upstream side of the site where the conjugate is immobilized, or may be included in the entirety of the conjugate pad.

As a material suitable for the conjugate pad, non-woven fibers such as paper, cellulose mixture, nitrocellulose, polyester, acrylonitrile copolymer, glass fiber, or rayon may be cited, but not limited thereto. Glass fiber mats are suitable.

If necessary, the conjugate pad may contain a "control reagent" for ensuring the reliability of immunochromatography, for example, an antibody labeled with a labeling substance and not reacting with a sample component, or a highly antigenic protein such as KLH (keyhole limpet hemocyanin) labeled with a labeling substance. These control reagents are components (substances) which are considered to be unlikely to be present in the sample, and may be appropriately selected.

(3rd Pad)

In the present invention, 3rd Pad can be configured for the following purposes: among reaction components of the sample and the detection reagent, components unnecessary for detection of the object to be detected can be removed, and components necessary for the reaction can be smoothly developed on an insoluble film to which an antibody is immobilized.

For example, blood cells, insoluble disrupted blood cells, and the like are unwanted components to be detected and are desired to be removed. In addition, the 3rd Pad can also have the following additional effects at the same time: among aggregates produced by the antigen-antibody reaction, aggregates that are too large to move on the antibody-immobilized membrane and that do not develop smoothly are removed in advance. As the 3rdPad, any substance and form that allows components of the liquid and the object to be detected to pass through are also included.

As specific examples, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, pulp, fabric, and the like are included, but not limited thereto.

In the present invention, it is desirable to use a blood cell separation membrane in order to more reliably separate and remove blood cells that have not been completely removed only by the erythrocyte agglutinating agent and the sample pad.

(fixation of antibody to insoluble Membrane)

The fixation of the antibody against the analyte in the immunochromatographic reagent of the present invention to the insoluble membrane can be carried out by a known method. For example, in the case of the flow-through method, the antibody is adjusted to a predetermined concentration, and a predetermined amount of the liquid is applied to the insoluble film in a dot form or a specific sign form such as + form. In this case, in order to ensure the reliability of the immunochromatography, a protein or a compound capable of binding to the conjugate is usually immobilized at a position different from the position of the antibody against the detection target, and used as a "control line". Alternatively, the antibody against the control reagent may be immobilized at a position different from the position of the antibody against the detection target, thereby forming a "control line".

In the case of the side stream, the following takes place: the insoluble film is coated in a linear form with the antibody adjusted to a predetermined concentration by using a device or the like having a mechanism capable of moving in a horizontal direction while discharging the liquid from a nozzle at a constant speed. In this case, the concentration of the antibody is preferably 0.1 to 5mg/mL, more preferably 0.5 to 3 mg/mL. The amount of antibody fixed to the insoluble membrane can be optimized by adjusting the amount of coating applied to the insoluble membrane in the case of the flow method, and by adjusting the discharge speed from the nozzle of the device in the case of the side flow method. Particularly, in the case of the lateral flow type, it is preferably 0.5 to 2. mu.L/cm. In the present invention, "flow-through membrane analysis" refers to a mode in which a sample liquid or the like is developed while passing through an insoluble membrane perpendicularly, and "lateral flow membrane analysis" refers to a mode in which a sample liquid or the like is developed while moving parallel to an insoluble membrane.

In the present invention, the position of the insoluble membrane coated with the antibody against the target may be arranged as follows in the case of the lateral flow: the above-mentioned detection reagents were developed from the conjugate pad based on capillary phenomenon, and passed through the wires coated with the respective antibodies in sequence. It is preferable to arrange the line coated with the antibody against the detection object at the upstream side and the line coated with the control antibody at the downstream side. In this case, the distance between the lines is desirably a sufficient distance to enable signal detection of the labeling substance. In the case of the flow scheme, the position of application of the antibody to the target may be arranged so that the signal of the label can be detected.

The antibody solution to be coated on the insoluble membrane can be prepared by using a predetermined buffer solution. Examples of the buffer solution include: phosphate buffer, Tris buffer, Good's buffer and the like commonly used buffers. The pH of the buffer is preferably in the range of 6.0 to 9.5, and may be appropriately set according to the properties of the antibody to be used. For example, in the case of the anti-H-FABP monoclonal antibody described later, a buffer solution of pH7.2 can be used. The buffer solution may further contain salts such as NaCl, stabilizers or preservatives such as sucrose, preservatives such as ProClin, and the like. The salts include salts such as NaCl that are included for adjusting the ionic strength, and salts such as sodium hydroxide that are added in the pH adjustment step of the buffer solution. After the antibody is fixed to the insoluble membrane, a commonly used blocking agent may be further coated in a solution or vapor state at a position other than the antibody fixing site to block the antibody.

In the present specification, as described above, the insoluble film to which the antibody is fixed may be referred to as an "antibody-fixed film".

(insoluble film)

In the present invention, as the insoluble film (hereinafter, may be simply referred to as a film), a film of any material may be used. Examples thereof include: polyethylene, polyethylene terephthalate, nylon, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, and the like, but not limited thereto. Specific examples thereof include glass fiber filters and cellulose filters sold by Merck & co, inc, eastern filter paper company, Whatman, inc. Further, by appropriately selecting the pore size and structure of the insoluble membrane, the flow rate of the immune complex between the colloidal gold-labeled antibody and the target substance in the membrane can be controlled. The amount of the labeled antibody bound to the above antibody immobilized on the membrane can be adjusted by controlling the flow rate in the membrane, and therefore it is desirable to optimize the pore size and structure of the membrane in consideration of the combination with other constituent materials of the test strip for immunochromatography of the present invention.

(absorbent pad)

In the present invention, the absorbent pad means: the developed liquid-absorbent site of the sample is controlled by absorbing the sample moving through the insoluble film. In the case of the lateral flow method, the reagent strip may be disposed at the most downstream side, and in the case of the flow method, the reagent strip may be disposed, for example, below the antibody-immobilizing film. As the absorbent pad, for example, filter paper may be used, but not limited thereto. 740-E, et al, by Whatman, Inc. are preferably used.

(detection device)

The immunochromatographic test strip of the present invention can be stored and mounted in an appropriate container (housing) in consideration of the size of the test strip, the method and position of adding a sample, the position of fixing an antibody on an antibody-fixing film, the method of detecting a signal, and the like, and such a stored and mounted state is referred to as "device".

(others)

In the specification, the "insoluble membrane" is sometimes referred to as "solid phase", and the state where an antigen or an antibody is supported on the insoluble membrane or after the support by a physical or chemical method is sometimes referred to as "immobilization", "solid phase", "sensitization", or "adsorption".

(sample)

In the detection method of the present invention, the "sample" containing the detection target is a liquid containing a particulate component having a large number of negative charges on the surface, and examples of the biological sample include a sample containing red blood cells, and particularly include: whole blood, red blood cells separated by centrifugation or the like, plasma, and the like.

In addition, the blood subject includes the following subjects: in blood collection, a subject collected with a blood collection tube to which an anticoagulant such as EDTA or heparin is added (hereinafter, may be simply referred to as an EDTA-containing subject, a heparin-containing subject, or the like).

According to the present invention, the following effects are also exerted: the problem that the measurement value of an EDTA-containing sample is lower than that of a heparin-containing sample in immunochromatography is solved, and accurate measurement can be performed regardless of the type of sample. Further, according to the additive of the present invention, an effect of increasing the detection sensitivity is also obtained. Therefore, by selecting the concentration and the type of the additive to be desired concentrations and types, the detection sensitivity of the immunochromatography apparatus can be controlled.

(detection target)

Examples of the object to be detected of the present invention include substances present in a biological sample such as blood (whole blood), red blood cells, serum, plasma, urine, saliva, or sputum, for example, inflammation-related markers such as CRP (C-reactive protein), IgA, IgG, and IgM; coagulation and fibrinolysis markers such as fibrin degradation products (e.g., D-dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex), and the like; circulation-related markers such as oxidized LDL, BNP (brain natriuretic peptide), H-FABP (cardiac fatty acid binding protein); metabolic-related markers, such as adiponectin; tumor markers, such as CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA19-9, CA125, PSA (prostate specific antigen); infection-related markers such as HBV (hepatitis B virus), HCV (hepatitis C virus), chlamydia trachomatis, neisseria gonorrhoeae; allergen-specific IgE (immunoglobulin E); a hormone; drugs, and the like. Among these, D dimer, CRP, BNP, H-FABP, etc., which are high in call sound and intended to use whole blood as a sample, are more preferable.

(antibody used in the present invention)

The antibody against the detection target used in the present invention is not limited to any specific method as long as it specifically reacts with the detection target, and may be a polyclonal antibody or a monoclonal antibody. Usually, hybridomas producing such antibodies are prepared by cell fusion of spleen cells of an animal immunized with a test object as an immunogen with myeloma cells of the same species (myelomas) according to the method of Kohler and Milstein (see Nature, volume 256, page 495 (1975)).

Here, in the measurement method for detecting an object to be detected by forming a so-called sandwich, in the case where the antibody used is a monoclonal antibody, the relationship between the antibody for label fixation (first antibody) and the antibody for insoluble membrane fixation (second antibody) is: when the epitope of the first antibody is monovalent, an epitope different from that of the first antibody is used as the epitope of the second antibody; when the epitope of the first antibody is multivalent, the epitope of the second antibody may be the same as or different from that of the first antibody.

In example 1 described later, an anti-H-FABP monoclonal antibody was used. The method for producing the anti-H-FABP monoclonal antibody used in the present invention is as follows, but the present invention is not limited thereto, and a commercially available anti-H-FABP monoclonal antibody may be used. Examples of commercially available anti-H-FABP monoclonal antibodies include: clone #5B5, #10E1, etc. of Hytest corporation; clone # M79188, #79189, etc. of Fitzgerald (note that, for convenience, a monoclonal antibody is sometimes represented by the clone name of a hybridoma producing the antibody.

(preparation example of anti-H-FABP monoclonal antibody)

(1) Preparation of hybridomas

Purified human H-FABP (manufactured by Hytest) dissolved in PBS was used as an immunogen. This immunogen was mixed and emulsified with Freund's complete adjuvant (Wako pure chemical industries, Ltd.) in an amount of 1: 1 to prepare an emulsion having a H-FABP concentration of 0.5mg/mL, and 100. mu.L of this emulsion was administered subcutaneously to 6-week-old female BALB/C mice. Then, 100. mu.L of an emulsion containing 0.2mg/mL of H-FABP was additionally administered for 3 times in 2 and a half months, and after 10 days from the 3rd additional administration, 100. mu.L of human purified H-FABP dissolved in PBS at 0.2mg/mL was subcutaneously administered. After 3 days, spleen, mouse neck lymph node and iliac lymph node were removed, and spleen and lymph node cells obtained were mixed with myeloma cells SP2/O-Ag14 at a ratio of 6: 1, and subjected to cell fusion in the presence of 50% polyethylene glycol 1540 (manufactured by Wako pure chemical industries, Ltd.). Fused cells were 2.5X 10 in terms of spleen cells ⁶Each of the cells was suspended in HAT medium in an amount of 0.2mL per well and dispensed into a 96-well plate (CORNING). It was purified at 5% CO ₂After culturing at 37 ℃ in an incubator for about 1 week, cell lines producing antibodies reactive with H-FABP were selected by ELISA for culture supernatants in wells in which hybridomas gradually grew. Specifically, IgG in each culture supernatant was immobilized on a microplate (manufactured by NUNC) via a goat anti-mouse IgG (Fc) antibody (manufactured by JACKSON), and then reacted with H-FABP. Then, the reaction mixture was reacted with a biotin-labeled anti-H-FABP rabbit polyclonal antibody (manufactured by Proteitech Group Co., Ltd.), and further reacted with peroxidase-labeled streptavidin (manufactured by PIERCE Co., Ltd.). Then, a peroxidase substrate solution containing o-phenylenediamine (manufactured by Tokyo chemical Co., Ltd.) was added to develop a color, 1.5N sulfuric acid was added to stop the color development, and then the resultant mixture was measured with a microplate reader (abs.492nm) to select a hybridoma exhibiting high reactivity to H-FABP. For the selected hybridomas, cloning was performed using the limiting dilution method, and 10 hybridomas producing anti-H-FABP monoclonal antibodies were established.

(2) Preparation of monoclonal antibodies

For female BALB/C mice 12 weeks old injected with pristane 0.5mL in 2 weeks anterior abdominal cavity, the cell number is 0.2X 10 ⁵The hybridoma obtained in (1) above was administered intraperitoneally in the same amount as the above. Ascites was collected about 14 days later and centrifuged to obtain a supernatant. The supernatant was mixed with an equal amount of a buffer for adsorption (3mol/L NaCl-1.5mol/L Glycine-NaOH, pH8.5), followed by filtration. The filtrate was passed through a protein A column (manufactured by GE Healthcare) equilibrated with an adsorption buffer, and after adsorbing the antibody in the filtrate to the column, the antibody was eluted from the column with 0.1mol/L citric acid buffer (pH3.0), thereby purifying the anti-H-FABP monoclonal antibodies (Clone #87203 and Clone # 87212).

(measurement)

The signal from the colloidal gold may be quantified by a known method, and the absorbance or the intensity of reflected light may be measured. The concentration of the target can also be measured by extrapolating the amount of change in the absorbance or reflected light intensity to a standard curve of a sample having a known concentration.

(detection method by immunochromatography)

The detection method by immunochromatography of the present invention is a method having at least the following steps (a) to (E), and typically is a detection method using the above-described test strip for immunochromatography.

(A) A step of supplying a sample to a sample supply portion of the test strip,

the test strip has a film composed of a porous body provided with at least a sample supply portion, an expansion portion, and a detection portion,

an antibody (conjugate) labeled with colloidal gold and directed to an object to be detected is held in a local part of the developed part so as to be eluted, and a detection part to which an immobilized antibody is fixed is provided in a local part of the developed part and downstream of the conjugate holding part;

(B) bringing the polybrene into contact with the sample at or upstream of the sample supply part to aggregate blood-derived components in the sample

(C) Separating and removing the aggregate obtained in the step (B) from the sample

(D) A step of bringing the sample component separated and removed of the agglutinate obtained in the step (C) into contact with a colloidal gold-containing conjugate in the presence of an additive having an ability to inhibit the agglutination of the conjugate due to polybrene

(E) A step of detecting the complex of the target substance and the conjugate in the sample component obtained in the step (D) in the detection section