JP2010122002A - Antibody detecting method and reagent kit used therein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody detecting method further enhanced in sensitivity as compared with a conventional method for detecting an antibody in a biosample, and a reagent kit used therein. <P>SOLUTION: The antibody detecting method includes a step for forming a composite of the antibody being a detection target in the biosample sampled from an examinee, an antigen to which a polyethylene glycol chain is added and which is bondable to a solid phase, and the solid phase, a step for bonding a substance which is labelled by a label substance and can be bonded to the antibody to the antibody forming the composite to label the composite, and a step for detecting the labelled composite. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting an antibody with improved sensitivity and a reagent kit used in the method.

  As an immunological technique for detecting an antibody in a sample, an antigen against an antibody to be detected is immobilized on a solid phase, an antigen-antibody complex is formed on the solid phase, and then the immobilized antibody Is labeled with a labeling substance, and the labeled amount is detected. In this case, a synthetic peptide, a gene recombinant protein, or the like is used as the antigen.

  Here, when a synthetic peptide is immobilized on a solid phase as an antigen, if its molecular weight is small, the reactivity with an antibody to be detected may be lowered due to steric hindrance caused by a blocking agent on the solid phase. In such a case, a synthetic peptide and a carrier protein such as BSA are combined and used as a composite antigen (for example, JP-A-2005-257700 (Patent Document 1)), or an appropriate linker such as polylysine. (For example, JP-A-6-319570 (Patent Document 2)) increases the reactivity with an antibody.

In addition, adding a polyethylene glycol chain to a protein is conventionally known as one of protein modification methods. However, it is known that a protein to which a polyethylene glycol chain is added is not easily recognized by the host immune mechanism, that is, its antigenicity is reduced (for example, JP-A 2006-169263 (Patent Document 3)). Depending on the modification method, the three-dimensional structure of the antigen may change, resulting in a change in antigenicity.
JP 2005-257700 A JP-A-6-319570 JP 2006-169263 A

  It is an object of the present invention to develop a method with improved sensitivity as compared with conventional antibody detection methods.

In order to solve the above problems, the present inventors have found that the sensitivity of the assay is improved when an immunoassay is performed using a synthetic peptide antigen to which a polyethylene glycol chain is added.
As described above, it is known that a protein to which a polyethylene glycol chain has been added has reduced antigenicity, but the present inventors unexpectedly added a polyethylene glycol chain to the antigen. As a result, the inventors have found that the reactivity to antibodies in a biological sample is improved, thereby completing the present invention.

  Therefore, the present invention provides a step of forming a complex between a solid phase and an antibody to be detected in a biological sample, an antigen against the antibody to which a polyethylene glycol chain has been added and which can be bound to the solid phase; Detection of an antibody comprising the steps of binding a substance labeled with a labeling substance and capable of binding to the antibody to the antibody forming the complex, and labeling the complex; and detecting the labeled complex Provide a method.

  The method of the present invention can provide a method for detecting an antibody with higher sensitivity.

  The antibody detection method of the present invention is based on an immunoassay based on the binding between an antibody to be detected and an antigen against the antibody to which a polyethylene glycol chain has been added and which can be bound to a solid phase. By separating the complex obtained by bringing the above antigen into contact with the above antibody that can be contained in the biological sample from the biological sample using a solid phase, the complex, that is, the antibody bound to the antigen Can be detected.

The antigen used in the present invention may be any of those recovered from nature, those prepared by recombinant genetic engineering techniques, or those chemically synthesized. Methods for recovering antigens from nature, methods for producing them by recombinant genetic engineering techniques, and methods for chemical synthesis are known per se.
For example, when chemically synthesizing an antigen, either a solid phase synthesis method or a liquid phase synthesis method may be used. Moreover, you may synthesize | combine using a commercially available peptide synthesizer.
In addition, as an antigen used in the present invention, a hepatitis antigen is preferable, and an antigen of non-A non-B non-C non-D non-E non-G hepatitis (non-AG hepatitis) is particularly preferable. Here, the non-AG hepatitis antigen preferably includes at least the amino acid sequence at positions 11 to 22 of SEQ ID NO: 1, and more preferably includes at least the amino acid sequence at positions 1 to 22 of SEQ ID NO: 1.

The “polyethylene glycol chain” added to the antigen against the antibody to be detected in the method of the present invention preferably contains at least four ethylene oxide units. In the polyethylene glycol chain, ethylene oxide units (— (CH ₂ —CH ₂ —O) —) may be directly bonded, or a linker structure may be interposed between these ethylene oxide units. Examples of the linker structure include -RCONH- group and -NHCOR- group (R is an alkylene group having 1 to 3 carbon atoms).
A linker structure may be interposed between the polyethylene glycol chain and the antigen. Examples of such a linker structure include an alkylene group having 1 to 3 carbon atoms.

The upper limit of the number of ethylene oxide units contained in the polyethylene glycol chain is usually 12, but more preferably 8, although it depends on the type of antigen used.
The number of ethylene oxide units added to the antigen can be confirmed by an electrospray / ionization mass spectrometry (ESI-MS) meter, for example, API2000 manufactured by Applied Biosystems.

Addition of the polyethylene glycol chain to the antigen for the antibody to be detected can be performed by a method known per se.
For example, a polyethylene glycol chain can be added to an antigen using the same principle as a chemical peptide synthesis reaction. That is, a compound having a polyethylene glycol chain that protects either the amino group or the carboxyl group and activates the other is used in place of the amino acid that is condensed during peptide synthesis, and is condensed at the end of the peptide chain of the antigen. Can be made. More specifically, an appropriate protecting group such as 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Bz), p-biphenyl is present at the N-terminus of the antigen. Carboxylic acid derivatives having a polyethylene glycol chain in which the amino group is protected with isopropyloxycarbonyl and the carboxyl group is activated, such as Fmoc-8-amino-3,6-dioxaoctanoic acid, Boc-8-amino-3 , 6-dioxaoctanoic acid, Fmoc-11-amino-3,6,9-trioxaundecanoic acid, Boc-11-amino-3,6,9-trioxaundecanoic acid, etc. are reacted, and this reaction is desired It is possible to use a method that repeats until a polyethylene glycol chain having a length of 1 is obtained.
The carboxylic acid derivative having a polyethylene glycol chain and having the amino group protected as described above is commercially available, and, for example, mini-PEG (trademark) manufactured by Peptides International can be used.

In addition, for example, a polyethylene glycol chain can be added to the antigen via a group reactive with an amino group or thiol group in the antigen. Examples of the group reactive with the amino group include an N-hydroxysuccinimidyl (NHS) group, and examples of the group reactive with the thiol group include a maleimide group.
Polyethylene glycol chains having these reactive groups are commercially available. For example, methyl-PEO ₄ -NHS ester, methyl-PEO ₈ -NHS ester, methyl-PEO ₁₂ -NHS ester, etc., manufactured by PIERCE can be used.

  The polyethylene glycol chain may be added to any position of the amino acid sequence of the antigen as long as it does not impair the antigenicity of the antigen to the antibody to be detected, but is added to the N-terminal or C-terminal. It is preferable.

  As a mode for allowing the antigen to bind to the solid phase, a mode known in the art can be used. Preferably, a solid phase binding site is added to the antigen, a binding substance capable of binding to the solid phase binding site is immobilized on the solid phase, and the antigen is bound to the solid phase by binding the solid phase binding site to the binding substance. Can be combined.

The solid-phase binding site and the binding substance are not particularly limited as long as they are a combination of substances that can specifically bind under the reaction conditions of the method of the present invention. Examples of these combinations include biotin and avidin, hapten and anti-hapten antibody, nickel and histidine tag, glutathione and glutathione-S-transferase, and the like.
Examples of the hapten and anti-hapten antibody include DNP and anti-DNP antibody. Preferably, it is a combination of biotin and avidins. More preferably, the solid phase binding site contains biotin and the binding substance is avidin. Each substance of the combination of the solid phase binding site and the binding substance may be bound to the antigen or the solid phase, and is not particularly limited. In the present specification, “avidins” means including avidin and streptavidin.

  Each of the polyethylene glycol chain and the solid phase binding site may directly bind to the antigen against the antibody to be detected, but the solid phase binding site may bind to the polyethylene glycol chain bound to the antigen. preferable. By setting it as such a structure, the antibody in a biological sample can be couple | bonded with a solid phase through the antigen with respect to the said antibody, and a polyethyleneglycol chain.

  The coupling between the polyethylene glycol chain and the solid phase binding site can be performed by a method known per se according to the type of the solid phase binding site. For example, the solid phase binding site and the polyethylene glycol chain can be bound by performing a reaction that forms an amide bond between the end of the solid phase binding site and the end group of the polyethylene glycol chain.

In addition, a method for binding a solid phase binding site to the antigen when the solid phase binding site directly binds to an antigen against the antibody to be detected is known in the art. For example, when the solid-phase binding site contains biotin, the carboxyl group of biotin is converted into a condensing agent (for example, HBTU (1- [bis (dimethylamino) methylene] -1H-benzotriazolium- 3-oxide hexafluorophosphate), carbodiimide, etc.) can be used to condense to the N-terminal amino group of the antigen.
In addition, biotin can be bound to the antigen via a group reactive with an amino group or thiol group in the antigen. Examples of the group reactive with an amino group include an NHS group, and examples of the group reactive with a thiol group include a maleimide group.

As the solid phase, those commonly used in the art can be used. Examples of the solid phase material include latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, and polyglycidyl methacrylate. , Polymer materials such as acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone; agarose; gelatin; erythrocytes; inorganic materials such as silica gel, glass, inert alumina, and magnetic materials . You may combine these 1 type, or 2 or more types.
Examples of the shape of the solid phase include a microtiter plate, a test tube, beads, particles, and nanoparticles. Examples of the particles include magnetic particles, hydrophobic particles such as polystyrene latex, copolymer latex particles having hydrophilic groups such as amino groups and carboxyl groups on the particle surface, erythrocytes, and gelatin particles.

More preferably, the solid phase is a magnetic particle.
The magnetic particles are particles containing a magnetic material as a base material. Such magnetic particles are known in the art, and those using, for example, Fe ₂ O ₃ and / or Fe ₃ O ₄ , cobalt, nickel, ferrite, magnetite or the like as a substrate are known. For the purpose of binding proteins or the like to the surface of the magnetic particles, a substrate whose surface is coated with a polymer or the like is more preferable.

Methods for immobilizing the above binding substance on a solid phase are known in the art. The immobilization can be performed by, for example, a physical adsorption method, a covalent bond method, an ionic bond method, or a combination thereof.
When the binding substance is avidin, for example, avidin can be directly immobilized on the solid phase by physical adsorption. Alternatively, the avidin can be immobilized on the solid phase by a method of binding the avidin to a solid phase to which avidin can be bound, for example, biotin. Avidins can also be immobilized on a solid phase by the method described in JP-A-2006-226689.
Further, the solid phase to which avidins are bound can be purchased from, for example, JSR Corporation or Dynal Biotech.

In the method of the present invention, a complex of an antibody that can be contained in a biological sample, the above-described antigen, and a solid phase is formed. In order to form the complex, the antibody, the antigen, and the antigen may be brought into contact with each other so that the solid phase is bound. That is, a complex can be formed by mixing an antibody, an antigen and a solid phase.
The contact order of the biological sample, the antigen, and the solid phase is not particularly limited, and the solid phase may be added after mixing the biological sample and the antigen, or the antigen and the solid phase are mixed and then added to the biological sample. Alternatively, the antigen may be added after mixing the biological sample and the solid phase. Further, the biological sample, the antigen, and the solid phase may be contacted at the same time. Since the reaction between the antigen and the antibody is likely to proceed, the solid sample is added after mixing the biological sample and the antigen, that is, after the antibody and the antigen form a complex, the antigen in the complex Is preferably bound to the solid phase.

  The biological sample is a sample collected from a human subject to be examined, and includes blood samples such as whole blood, serum, and plasma.

  In the method of the present invention, the antigen against the antibody to be detected is preferably reacted in excess with the antibody that can be contained in the biological sample, depending on the type of antigen used. More specifically, the concentration of the antigen in the reaction may be 0.1 to 10 μg / ml, preferably 0.3 to 3 μg / ml, and particularly preferably 0.5 to 2 μg / ml.

The method of the present invention can be performed under the reaction conditions of a conventional immunoassay. Such conditions are preferably pH 6 to 10 and 30 to 45 ° C.
Further, the time for contacting the biological sample, the antigen, and the solid phase is not particularly limited, but is usually 1 to 5 minutes.

  As described above, the antigen-antibody complex bound to the solid phase can be separated from the biological sample by collecting the solid phase. It is preferable to perform washing simultaneously with the separation to remove antigens and antibodies not forming a complex. Cleaning can be performed using a cleaning solution known in the art.

The antibody bound to the antigen separated from the biological sample as described above is detected using a substance that is labeled with a labeling substance and that can bind to the antibody. That is, an antibody bound to a solid phase via an antigen is labeled with a labeling substance that can bind to the antibody. And what is necessary is just to detect the labeled antibody.
The substance that can bind to the antibody is a substance that can bind to the human antibody, and an antibody against the human antibody is preferably used. Antibodies against human antibodies include antibody fragments and derivatives thereof. Specific examples include Fab fragments, F (ab ′) fragments, F (ab) ₂ fragments, and sFv fragments. Antibody classes can include, but are not limited to, IgG, IgM, and the like.

The labeling substance is not particularly limited as long as it can be used in a normal immunoassay, and examples thereof include enzymes, fluorescent substances, and radioisotopes. Examples of the enzyme include alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, and acid phosphatase. Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), and luciferin. Examples of the radioisotope include ¹²⁵ I, ¹⁴ C, and ³² P.

The measurement of the labeling substance can be performed according to a method known in the art, and can be appropriately selected depending on the type of the labeling substance.
For example, when the labeling substance is an enzyme, the labeling substance can be measured by performing color development or luminescence reaction using a substrate for the enzyme. For example, if the enzyme is alkaline phosphatase, the substrate may be CDP-star® (4-chloro-3- (methoxyspiro {1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo). [3.3.1.1 ^3,7 ] decan} -4-yl) phenyl phosphate disodium), CSPD® (3- (4-methoxyspiro {1,2-dioxetane-3,2- Chemiluminescent substrates such as (5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decan} -4-yl) phenyl phosphate disodium); p-nitrophenyl phosphate, 5-bromo-4- A chromogenic substrate such as chloro-3-indolyl-phosphate (BCIP), 4-nitroblue tetrazolium chloride (NBT), iodonitrotetrazolium (INT) can be used.
Luminescence or color development by these substrates can be measured with a luminometer or spectrophotometer.

The present invention also provides an antibody detection reagent kit comprising an antigen to an antibody to be detected, to which a polyethylene glycol chain is added and which can be bound to a solid phase, and a solid phase.
The antigen and the solid phase may be contained in the same reagent or in different reagents. When the antigen and the solid phase are contained in the same reagent, the antigen and the solid phase may be contained in the reagent in a state of being bound in advance. More preferably, the antigen and the solid phase are contained in separate reagents.

  The reagent kit preferably further includes a substance that is labeled with the labeling substance and that can bind to the antibody in the biological sample. The substance is preferably contained in a reagent other than the antigen and the solid phase.

  Each reagent contained in the reagent kit of the present invention may be a liquid, or may be a solid for use with water added at the time of use.

  The reagent in the liquid form may be any reagent in which at least each of the above components is dissolved in an appropriate solvent. Suitable solvents include water, pH 6-10 buffer and the like. As the buffer, phosphate buffered saline (PBS), triethanolamine hydrochloride buffer (TEA), Tris-HCl buffer (Tris-HCl), 2-morpholinoethanesulfonic acid (MES), etc. should be used. Can do. The buffer may contain a known additive such as a surfactant, a preservative, and serum protein.

  The reagent in the above solid form can be obtained by subjecting an appropriate solvent in which the above components are dissolved to lyophilization or the like.

  The amount of the antigen contained in the reagent kit of the present invention is such that when the antibody that can be contained in the biological sample reacts with the antigen, the amount is excessive with respect to the antibody that can be contained in the biological sample. Good. That is, the concentration at which the antibody reacts with the antigen is 0.1 to 10 μg / ml, preferably 0.3 to 3 μg / ml, and particularly preferably 0.5 to 2 μg / ml.

The following examples illustrate the invention in more detail, but the invention is not intended to be limited by the following examples.
<Production of non-AG hepatitis antigen to which biotin is bound and a polyethylene glycol chain containing four ethylene oxide units is added>
A non-AG hepatitis A antigen having the amino acid sequence of positions 1 to 22 of SEQ ID NO: 1 was synthesized by Fmoc solid phase method using a peptide synthesizer Model 433A (Applied Biosystems) according to the manufacturer's instructions. . Specifically, it is as follows.
According to the instruction manual of the above peptide synthesizer, a commercially available Fmoc-phenylalanine-Wang resin is dispensed into a reaction vessel, the first amino acid Fmoc-serine at the C-terminal of the polypeptide to be synthesized, and HBTU (1 -[Bis (dimethylamino) methylene] -1H-benzotriazolium-3-oxide hexafluorophosphate), HOBt (1-hydroxybenzotriazole) as additive, NMP (N-methyl-2-pyrrolidone) as solvent Were added to the reaction vessel and allowed to react. To the obtained resin-dipeptide, DMF containing 20% by weight piperidine was added, and after removing the Fmoc group, the second amino acid Fmoc-proline was reacted in the same manner as described above. This was repeated, and the Fmoc-amino acids from the C-terminal to the 1st to 22nd were reacted in order to obtain a non-AG hepatitis antigen.

  Next, the same reaction was repeated twice using Fmoc-mini-PEG (Fmoc-8-amino-3,6-dioxaoctanoic acid; manufactured by Peptides International) instead of amino acid, and non-AG Two molecules of mini-PEG were bound to the N-terminus of hepatitis B antigen.

Furthermore, biotin-2xmini-PEG-antigen was bound in this order from the N-terminal by reacting with biotin and the above-mentioned condensing agent, additive and solvent, and reacting in this order. Hepatitis antigen was obtained.
The biotin-PEG-added non-AG hepatitis antigen was cleaved from the resin and purified by removing the biotin-PEG-added non-AG hepatitis antigen bound to the resin with a deprotection solution (87.5% by volume trifluoromethane). Acetic acid, 5% by volume thioanisole, 2.5% by volume ethanediol, 5% by volume m-cresol) for 2 hours at room temperature to cleave the antigen from the resin and 0.1% by weight by reverse phase chromatography on an ODS column. This was done by eluting with a linear gradient of acetonitrile solution containing trifluoroacetic acid.

<Production of non-AG hepatitis antigen conjugated with biotin>
In the same manner as described above, a non-AG hepatitis antigen having the amino acid sequence of positions 1 to 22 of SEQ ID NO: 1 was synthesized.
By reacting biotin with the above condensing agent, additive and solvent, biotin-added non-AG hepatitis antigen bound with biotin was obtained.
Cutting out from the resin and purification were carried out in the same manner as described above.

Example 1
Hepatitis patient blood sample (whole blood) 10μl, 120μl of 1st reagent (0.1M TEA, 0.1% BSA, pH7.6) containing 1μg / ml biotin-PEG added non-AG hepatitis antigen is added and mixed And incubated at 42 ° C. for 2.25 minutes.
Next, add 30 μl of a second reagent containing streptavidin magnetic particles as a solid phase (average particle size 2 μm, particle concentration 0.5%, 20 mM MES, 0.1% BSA, pH 6.5), and mix at 42 ° C. for 1.5 minutes. Incubated.
Magnetic separation was performed to separate the antigen-antibody complex bound to the solid phase.
Washing solution (0.01 M MES, pH 6.5, 0.1% sodium azide) 100-700 μl was added and stirred to perform magnetic separation. This washing operation was repeated three times.

  Next, as a substance capable of binding to the human-type antibody labeled with the labeling substance, a third reagent containing alkaline phosphatase (ALP) -labeled anti-human IgG antibody (4.55 ng / ml; 100 mM MES, 1 mM magnesium chloride, 0.1 mM ZnCl2, 0.15M NaCl, pH 6.5) 100 μl was added, mixed, and incubated at 42 ° C. for 2.75 minutes. The same washing operation as described above was performed three times.

To this, add 50 μl of a diluted solution consisting of 100 mM MES, 3 mM magnesium chloride hexahydrate, pH 6.5, mix, and then add 100 μl of luminescent substrate solution (CDP-Star, Applied Biosystems) as a substrate for ALP. The mixture was stirred and allowed to react at 42 ° C. for 5 minutes.
The obtained emission intensity counts are shown in Tables 1 and 2.

Comparative Example 1
In Example 1, the same procedure as Example 1 was repeated using biotin-added non-AG hepatitis antigen instead of biotin-PEG-added non-AG hepatitis antigen, and the luminescence intensity was measured. The obtained emission intensity counts are shown in Tables 1 and 2.

  From these results, when using an antigen to which a polyethylene glycol chain was added, compared to using an antigen to which a polyethylene glycol chain was not added, when using a non-AG hepatitis positive specimen, The luminescence intensity increased about 1.5-17 times. This increase in sensitivity was not seen when non-AG hepatitis negative specimens were used.

Example 2
In the first reagent used in Example 1, the first reagent (A), the first reagent (B), the first reagent (C), and the first reagent having different concentrations of biotin-PEG-added non-AG hepatitis antigens. (D) and the first reagent (E) were prepared. The concentration of biotin-PEG-added non-AG hepatitis antigen contained in each reagent is shown below.
First reagent (A): 0.1 μg / ml
First reagent (B): 0.3 μg / ml
First reagent (C): 1 μg / ml
First reagent (D): 3 μg / ml
First reagent (E): 10 μg / ml
The first reagent (C) is the same reagent as the first reagent used in Example 1.

  Using the first reagents (A) to (E), the count of luminescence intensity of five non-AG hepatitis positive specimens was measured in the same manner as in Example 1. Table 3 shows the ratio of the emission intensity count obtained with each reagent, with the emission intensity count obtained with the first reagent (C) being 100%. Moreover, the graph which shows the average value of the ratio of the count of the emitted light intensity obtained with each reagent is shown in FIG.

  As is clear from Table 3 and FIG. 1, the biotin-PEG-added non-AG hepatitis antigen contained in the first reagent can be used at a concentration of 0.1 to 10 μg / ml, but 0.3 to 3 μg / ml It became clear that it was preferable.

It is a graph which shows the average value of the ratio of the count of emitted light intensity obtained using 1st reagent (A)-(E).

Claims (11)

A method for detecting an antibody contained in a biological sample collected from a subject,
Forming a complex of a solid phase with an antibody to be detected in a biological sample, an antigen against the antibody to which a polyethylene glycol chain has been added and capable of binding to the solid phase;
A step of binding a substance labeled with a labeling substance and capable of binding to the antibody to the antibody forming the complex, and labeling the complex; and a step of detecting the labeled complex;
A method for detecting an antibody, comprising:   The method of claim 1, wherein the polyethylene glycol chain is a polyethylene glycol chain comprising at least 4 ethylene oxide units.   The method according to claim 1 or 2, wherein the antigen is a hepatitis antigen.   In the complex formation step, an antibody to be detected in a biological sample is contacted with an antigen against the antibody to form an antigen-antibody complex, and then the antigen-antibody complex is contacted with a solid phase, 4. The method according to any one of claims 1 to 3, wherein an antigen-antibody-solid phase complex is formed.   The method according to any one of claims 1 to 4, wherein the antigen is capable of binding to a solid phase by binding of biotin and avidins.   The method according to any one of claims 1 to 5, wherein the substance capable of binding to the antibody is an anti-human IgG antibody, and the labeling substance is an enzyme.   An antibody detection reagent kit comprising an antigen to an antibody to be detected, to which a polyethylene glycol chain is added and capable of binding to the solid phase, and a solid phase.   The reagent kit according to claim 7, wherein the polyethylene glycol chain is a polyethylene glycol chain containing at least four ethylene oxide units.   The reagent kit according to claim 7 or 8, further comprising a substance labeled with a labeling substance and capable of binding to the antibody.   The reagent kit according to any one of claims 7 to 9, wherein the antigen is a hepatitis antigen.   The reagent kit according to any one of claims 7 to 10, wherein the concentration of the antigen contained in the reagent is 0.3 to 3 µg / ml.

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