JP2019053060A - Immunoassay method for hepatitis B virus core antibody - Google Patents

Abstract

To provide an immunoassay method for hepatitis B virus core antibody capable of enhancing the detection sensitivity by suppressing non-specific reaction more than conventional method in immunity measurement of anti-HBc antibody.SOLUTION: Disclosed immunization measuring method of anti-HBc antibody in sample comprises a step of performing antigen-antibody reaction by contacting the sample with a reagent that includes the HBc antigen or anti-immunoglobulin antibody or an antigen-binding fragment thereof under a condition in which (1) nonionic surfactant and (2) at least one surfactant selected from amphoteric surfactants coexist with at least one surfactant. With this, the nonspecific reaction at the time of the immune measure of the anti-HBc antibody can be preferably suppressed, and further, suppress the background more than the conventional method.SELECTED DRAWING: None

Description

  The present invention relates to an immunoassay method for hepatitis B virus core antibody.

  The main body of hepatitis B virus (HBV) is a particle called “dane particle” which takes the shape of a double-structured spherical particle having a diameter of 42 nm. The surface of the Dane particle is covered with a surface antigen called HBs antigen, and the inside contains a double-stranded DNA containing HBc antigen (core antigen), HBe antigen, and a viral gene. It is known that a 27 nm core structure exists.

  These antigens and autoantibodies against the antigen have been targeted for the diagnosis of viral infection. In particular, antibodies against HBc antigen (generally called “HBV core antibody” or “anti-HBc antibody”) become positive from the early stage of HBV infection and remain positive for a long time after infection, thus widely detecting HBV infection It is used as a possible marker.

  However, there are cases where it cannot be detected despite the fact that the existing anti-HBc antibody measurement reagent is positive, and high sensitivity is required. As a means for measuring the anti-HBc antibody with high sensitivity, it is conceivable to increase the amount of the sample added to the reaction system. However, when the amount of the sample added to the reaction system is increased, the signal value of the positive sample increases, but at the same time, the background signal value due to the nonspecific reaction also increases, so the sensitivity cannot be increased.

  As a method for suppressing the nonspecific reaction of the immunoassay system of anti-HBc antibody, for example, Patent Document 1 describes a method of adding a reducing agent such as 2-mercaptoethanol or dithiothreitol to a pretreatment solution or a primary reaction solution. Has been. In Patent Document 1, it is assumed that false positives can be eliminated, and it is surmised that the nonspecific reaction is suppressed to some extent. However, the method using a reducing agent is not sufficient for suppressing the nonspecific reaction.

  Patent Document 2 discloses a technique of diluting a specimen with an IgM-containing aqueous solution as a means for solving the problems in the measurement of specific IgM antibodies that occur in the acute stage of infectious diseases. The disclosure is limited to the measurement of IgM antibodies, and mainly relates to a method for measuring IgM antibodies against hepatitis A virus (HAV). It is described that the HAV extract treated with an anionic surfactant is used as the HAV antigen reagent, and that a surfactant other than the anionic surfactant may be added to the measurement system. In the examples, an anti-IgM antibody-coated fine particle reagent containing 0.1% Tween20 is used. However, the purpose and effect of adding a surfactant other than the anionic surfactant in the measurement system are not described at all.

  In Patent Document 3, a nonionic surfactant having an HLB value of 12 to 19 is present in an antigen-antibody reaction system at a concentration of 1.5 to 6 w / w%, and an antigen-antibody reaction is performed. It is described that the non-specific interference is reduced and the measurement sensitivity and measurement accuracy of the immune reaction at low values are improved. According to the description of the examples, when the surfactant concentration in the antigen-antibody reaction system is a low concentration of less than 1.5%, neither the effect of lowering the background nor the effect of promoting the immune reaction is sufficient. In Examples, only the effect of NCC-ST-439 antigen in immunoassay is examined, and the immunoassay of antibody is not studied at all.

JP-A-1-321365 JP-A-8-5633 JP-A-6-66798

  An object of the present invention is to provide a means capable of further suppressing the nonspecific reaction and further enhancing the detection sensitivity in the immunoassay of an anti-HBc antibody.

  As a result of diligent research, the inventors of the present application conducted the immunoassay of the anti-HBc antibody under the condition that a predetermined nonionic surfactant or amphoteric surfactant coexists in the primary reaction system, thereby obtaining the surfactant. It was found that the non-specific reaction can be further suppressed and the detection sensitivity of the anti-HBc antibody can be enhanced as compared with the conventional similar technique used, and the present invention has been completed.

That is, the present invention is an immunoassay method for an anti-HBc antibody in a specimen, which comprises the following (1) and (2):
(1) Nonionic surfactant
(2) An antigen-antibody reaction by contacting a specimen containing a reagent containing an HBc antigen or an anti-immunoglobulin antibody or an antigen-binding fragment thereof under conditions where at least one surfactant selected from amphoteric surfactants coexists. A method is provided that includes:
The present invention also provides an anti-HBc antibody immunoassay kit comprising a solution containing at least one surfactant selected from (1) and (2) above and an HBc antigen. provide. Furthermore, the present invention is an immunoassay kit for an anti-HBc antibody, comprising an antigen reagent solution containing an HBc antigen, wherein the reagent solution is at least one interface selected from (1) and (2) above. A kit is provided containing an active agent. Furthermore, the present invention is an anti-HBc antibody immunoassay kit, comprising an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof, and an HBc antigen, wherein the antibody reagent solution is the above (1) And a kit containing at least one surfactant selected from (2).

  According to the present invention, a non-specific reaction at the time of immunoassay of an anti-HBc antibody can be satisfactorily suppressed, and the background can be further suppressed as compared with the conventional method. Therefore, the detection sensitivity at a low value is increased, and the background can be suppressed even when the amount of sample used is increased, so that highly sensitive measurement is possible.

The present invention is a method for measuring an anti-HBc antibody in a specimen by immunoassay. Under the conditions in which at least one surfactant selected from the following (1) and (2) coexists, an HBc antigen or anti-antibody An antigen-antibody reaction is performed by contacting a specimen with a reagent containing an immunoglobulin antibody or an antigen-binding fragment thereof.
(1) Nonionic surfactant
(2) Amphoteric surfactant

  The reagent containing the HBc antigen or anti-immunoglobulin antibody or antigen-binding fragment thereof (hereinafter sometimes referred to as “antigen / antibody reagent” for convenience) may be, for example, a reagent containing the HBc antigen. The antigen-antibody reaction described above is a reaction between a specimen and a reagent containing an HBc antigen (a reaction between an anti-HBc antibody in the specimen and an HBc antigen).

  The surfactant may be added to the specimen diluent, for example. That is, in this embodiment, a solution containing at least one surfactant is used, and an antigen-antibody reaction is performed by mixing a specimen, the solution, and an antigen / antibody reagent, thereby coexisting with the surfactant. The antigen-antibody reaction is carried out in (Aspect 1). The antigen / antibody reagent may not contain the surfactant. When the surfactant is also contained in the antigen / antibody reagent, it corresponds to the following embodiment 3.

  A surfactant may be included in the antigen / antibody reagent. That is, in one embodiment, an antigen-antibody reaction is carried out in the presence of a surfactant by mixing an analyte and an antigen / antibody reagent containing at least one surfactant as described above to perform an antigen-antibody reaction ( Aspect 2). When the sample diluent is used, the sample diluent may not contain the surfactant. When the above-mentioned surfactant is also contained in the specimen diluent, it corresponds to the following aspect 3.

  A surfactant may be included in both the antigen / antibody reagent and the sample diluent. That is, in one embodiment, by performing an antigen-antibody reaction by mixing a specimen, a solution containing the at least one surfactant, and an antigen / antibody reagent containing the at least one surfactant, Antigen-antibody reaction is performed in the presence of a surfactant (Aspect 3).

  The order of mixing the specimen, the specimen diluent, and the antigen / antibody reagent is not limited. After first mixing the sample diluent and the sample, at least a part of the diluted sample may be added to the antigen / antibody reagent, or the sample diluent and the sample may be added separately to the antigen / antibody reagent. . When added separately, the sample diluent and the sample may be added simultaneously or sequentially. When adding sequentially, either may be added to the antigen / antibody reagent first. It is common to first add the specimen diluent to the antigen / antibody reagent and then add the specimen, but the reverse is also possible. In this specification, the term “specimen diluent” is used for convenience, but it is intended to limit the scope of the present invention to an embodiment in which the sample diluent and the specimen are first mixed and then added to the reagent. It is not a thing.

  Examples of the nonionic surfactant (1) include ether type or ester ether type nonionic surfactants. A nonionic surfactant having an HLB (Hydrophilic-Lipophilic Balance) value of 10 to 20, for example, 11 to 20, 12 to 19, or 17 to 19 can be preferably used.

  Preferable examples of the ether type nonionic surfactant include polyoxyethylene alkylphenyl ether surfactants and polyoxyethylene alkyl ether surfactants.

  Preferable examples of the polyoxyethylene alkylphenyl ether surfactant include various surfactants known under the trade name of Triton (registered trademark) having an HLB value in the above range, particularly an HLB value of 17 to 19 Can be mentioned. Specific examples include polyoxyethylene (40) octylphenyl ether (trade name Triton X-405, HLB = 17.9), polyoxyethylene (30) octylphenyl ether (trade name Triton X-305, HLB = 17.3), polyoxy Ethylene (8) octylphenyl ether (trade name Triton X-114, HLB = 12.4), polyoxyethylene (10) octylphenyl ether (trade names Triton X-100, HLB = 13.5), and Triton X-705 Examples thereof include, but are not limited to, polyoxyethylene alkylphenyl ether surfactants having an HLB of 18.5 known by trade names.

  Preferable examples of the polyoxyethylene alkyl ether surfactant include those having an HLB value in the above range among various surfactants known under the trade name of Brij (registered trademark). Specific examples include polyoxyethylene (10) cetyl ether (trade name Brij C10, HLB = 13.0) and polyoxyethylene (20) cetyl ether (trade names Brij 58, HLB = 15.7). It is not limited.

  Preferable examples of the ester ether type nonionic surfactant include polyoxyethylene sorbitan fatty acid ester surfactants. Preferable examples of the surfactant include those having an HLB value in the above range among various surfactants known under the trade name of Tween (registered trademark). Specific examples include polyoxyethylene sorbitan monolaurate (trade name Tween 20, HLB = 16.7), polyoxyethylene sorbitan monopalmitate (trade name Tween 40, HLB = 15.6), polyoxyethylene sorbitan monostearate (trade name) Examples include, but are not limited to, Tween 60, HLB = 14.9) and polyoxyethylene sorbitan monooleate (trade names Tween 80, HLB = 15.0).

  As the amphoteric surfactant (2), a sulfobetaine surfactant can be preferably used. Of these, sulfobetaine surfactants having a linear alkyl group having 12 or more carbon atoms are preferred. Specific examples include N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C12APS), N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C14APS), N- Examples include, but are not limited to, hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate (C16APS).

  The liquid used as the base of the specimen dilution liquid is not particularly limited as long as it is an aqueous liquid. It may be a liquid containing components derived from human or animal blood. For example, sterilized water, a buffer having a buffer capacity of pH 4.0 to 11.0, and serum can be used. In Aspect 1 and Aspect 3, a liquid containing such a predetermined surfactant in a predetermined concentration in such an aqueous liquid may be used as the specimen dilution liquid. In the aspect 2, such an aqueous liquid may be used as it is as the sample diluent.

  The concentration of the surfactant in the antigen-antibody reaction system (the total concentration when two or more of the surfactants are used) is preferably 0.040 w / v% to 1.27 w / v%, For example, 0.040 w / v% to 1.0 w / v%, 0.040 w / v% to 0.80 w / v%, 0.040 w / v% to 0.50 w / v%, or 0.040 w / v% to 0.40 w / v% It may be. When used at about 0.40 w / v% or less, the reactivity of positive specimens can be maintained particularly high while suppressing nonspecific reactions well.

  Therefore, the sample diluent and the antigen / antibody reagent should contain the above surfactant so that the surfactant concentration in the above antigen-antibody reaction system is 0.040 w / v% to 1.27 w / v%. Is preferred. As in the following examples, assuming an immunoassay system in which 10 μL of a sample is mixed with 190 μL of a sample diluent, 10 μL of which is mixed with 50 μL of HBc antigen reagent to perform a primary reaction, the surfactant concentration in the primary reaction system is In the case of 0.040 w / v% to 1.27 w / v%, the concentration of the surfactant in the specimen diluent in Embodiment 1 is 0.25 w / v% to 8.02 w / v%, and in the antigen / antibody reagent in Embodiment 2 The surfactant concentration is 0.048 w / v% to 1.52 w / v%, and in Embodiment 3, the sample dilution is performed so that the surfactant in the primary reaction system is 0.040 w / v% to 1.27 w / v%. The surfactant concentration of the solution and the antigen / antibody reagent may be adjusted.

  When increasing the amount of sample used, adjust the surfactant concentration in the sample diluent and antigen / antibody reagent so that the surfactant concentration in the primary reaction system is 0.04 w / v% to 1.27 w / v%. It is preferable to do. When the amount of the sample used is about 10 times, for example, when 10 μL of the sample is mixed with 190 μL of the sample diluent, and 100 μL of the diluted sample is mixed with 50 μL of the HBc antigen reagent, the primary dilution is performed. The surfactant concentration of the solution may be 0.063 w / v% to 2.01 w / v%, and the surfactant concentration of the antigen / antibody reagent in embodiment 2 may be 0.12 w / v% to 3.81 w / v%. Then, the surfactant concentration of the sample diluent and the antigen / antibody reagent may be adjusted so that the surfactant in the primary reaction system is 0.040 w / v% to 1.27 w / v%. Alternatively, when 100 μL of the sample and 100 μL of the sample diluent are mixed and 10 μL of the sample is mixed with 50 μL of the HBc antigen reagent and the primary reaction is performed, the surfactant concentration in the sample diluent in Embodiment 1 is 0.48 w / v%. -15.2 w / v%, and the surfactant concentration of the antigen / antibody reagent in embodiment 2 may be 0.048 w / v%-1.52 w / v%. In embodiment 3, the surfactant in the primary reaction system is 0.040 w / v%. The concentration of the surfactant in the sample diluent and the antigen / antibody reagent may be adjusted so as to be w / v% to 1.27 w / v%.

  In the present invention, a salt may be used in combination with the surfactant. For example, the specimen diluent may further contain a salt. Further, the antigen / antibody reagent usually contains a salt of about 100 to 150 mM, but may contain a higher concentration of salt. A higher nonspecific reaction reducing effect can be obtained by combining a salt with the above-described predetermined surfactant. The salt is not particularly limited, and may be an inorganic salt or an organic salt. For example, alkali metal salts such as sodium chloride and potassium chloride can be preferably used, but are not limited thereto. A plurality of types of salts may be used in combination.

  When adding salt to the sample diluent, the salt concentration in the sample diluent (total concentration when multiple salts are used) may be 200 mM or more, for example, 250 mM or more, 300 mM or more, 350 mM or more 400 mM or more, 500 mM or more, or 800 mM or more. The upper limit is not particularly limited, but is usually about 2000 mM or less, for example, 1800 mM or less, or 1500 mM or less.

  When using a blood-derived sample such as whole blood, serum, or plasma, a salt derived from the sample is contained in the primary reaction solution. In the case of a blood-derived specimen, it generally contains about 150 mM of salt. As in the examples below, assuming an immunoassay system in which 10 μL of serum sample and 190 μL of sample diluent are mixed, 10 μL of this is mixed with 50 μL of HBc antigen reagent with a salt concentration of 150 mM, and a primary reaction is performed. When the salt concentration is 200 mM to 2000 mM, the salt concentration in the primary reaction system is 158 mM to 443 mM. Therefore, when the salt concentration in the case of using the salt in the present invention is expressed as the concentration in the primary reaction system, it may be 158 mM to 443 mM, for example, 170 mM to 443 mM, 180 mM to 443 mM, 190 mM to 443 mM, or 200 mM to 443 mM. possible. The salt concentration in the primary reaction system may be, for example, 420 mM or less, 400 mM or less, or 350 mM or less within the above range. In the present invention, the salt concentration in the primary reaction system may be achieved by increasing the salt concentration of the antigen / antibody reagent solution. That is, in the present invention, in order to reduce the nonspecific reaction, either one or both of the sample diluent and the antigen / antibody reagent solution have a salt concentration of 158 mM or more in the reaction system of the antigen-antibody reaction (primary reaction). It may contain a salt.

  In the present invention, it is not necessary to allow the predetermined surfactant to coexist in all steps of immunoassay. For example, as described later, when anti-HBc antibody is measured by a two-step sandwich immunoassay, surfactants coexist in the primary reaction system, but B / F separation and solid phase carrier washing are performed. In step, the surfactant is removed from the reaction system. In the subsequent steps, it is not essential to add the surfactant into the reaction system, and it may or may not be added. For example, the secondary reaction that is the second antigen-antibody reaction may be performed in the presence or absence of the surfactant described above.

  As is well known in the field of immunoassay, the primary reaction in the two-step sandwich method is a reaction between an antigen or antibody bound on a solid phase carrier and a specimen, and the secondary reaction is B / F separation. And a reaction between the solid phase after washing and the labeled antibody or labeled antigen. In the present invention, when the HBc antigen is immobilized, the reaction between the HBc antigen on the carrier and the diluted specimen is a primary reaction, and the reaction between the solid phase after B / F separation and washing and the labeled anti-immunoglobulin antibody is second. When the anti-immunoglobulin antibody is immobilized, the reaction between the anti-immunoglobulin antibody on the carrier and the diluted specimen is the primary reaction, and the reaction between the solid phase after B / F separation and washing and the labeled HBc antigen. Is a secondary reaction.

  Examples of the specimen used in the present invention include whole body blood, plasma, serum, urine, saliva, cerebrospinal fluid and other biological body fluids, liver tissue and the like, and particularly, whole blood, serum and plasma samples are preferable. However, it is not limited to these.

  The test individual is not particularly limited as long as it is a mammal, but is preferably a human. The test individual may be a patient suspected of having hepatitis B or a patient who has developed hepatitis (particularly hepatitis B).

  The anti-HBc antibodies to be measured include IgG type antibodies and IgM type antibodies. In the present invention, either one of IgG type antibody and IgM type antibody (for example, IgG type antibody) may be measured, or both may be measured. Typically, the anti-HBc antibody to be measured includes an IgG type antibody.

  The antibody measurement method itself is a well-known conventional method. The immunoassay can be classified by reaction mode, such as sandwich method, competition method, aggregation method, immunochromatography method, Western blot method, etc. When classified by label, radioimmunoassay, fluorescent immunoassay, enzyme immunoassay (EIA), biotin immunity There are measurements. Specific examples of antibody testing methods using antigens include, but are not limited to, EIA methods (ELISA, CLEIA (chemiluminescent enzyme immunoassay), Western blot, etc.), chemiluminescent immunoassays (CLIA method) , Aggregation method (latex aggregation method, etc.), complement binding reaction (CF) and the like. In the present invention, any technique may be used as long as it is an immunoassay that can specifically detect an anti-HBc antibody in a specimen. Although not particularly limited, the immunoassay in the present invention can be an immunoassay by a sandwich method, particularly a two-step sandwich method. In the present invention, the term “measurement” includes detection, quantification, and semi-quantification.

  Hereinafter, taking the CLEIA method based on the two-step sandwich method as an example, each step in the case of measuring an anti-HBc antibody in a human-derived specimen by the method of the present invention will be described more specifically.

  First, after diluting the human-derived sample with the sample diluent, the HBc antigen immobilized on the solid support is brought into contact with the diluted sample, or the sample diluent and the human-derived sample are separately and sequentially separated from the HBc antigen. By contacting (generally, the sample dilution solution is contacted first, but the sample may be first), the HBc antigen on the carrier is reacted with the anti-HBc antibody in the diluted sample (primary reaction). By this primary reaction, the anti-HBc antibody in the specimen is bound on the carrier via the HBc antigen. In aspect 1, the specimen diluent contains a surfactant, in aspect 2, the reagent containing the immobilized HBc antigen, and in aspect 3, both the specimen diluent and the immobilized HBc antigen reagent contain a surfactant. The reaction proceeds in the presence of a surfactant. After the reaction, B / F separation is performed and the separated carrier is washed. By the B / F separation and washing step, the predetermined surfactant contained in the specimen diluent or the immobilized HBc antigen reagent is removed from the reaction system.

  Next, a labeled anti-human immunoglobulin antibody is added to the system, and the anti-HBc antibody bound to the HBc antigen is reacted with the labeled anti-human immunoglobulin antibody (secondary reaction). By this secondary reaction, a complex of HBc antigen-anti-HBc antibody-labeled anti-human immunoglobulin antibody is formed on the solid phase carrier. Subsequently, B / F separation is performed, and the carrier is washed to remove unreacted labeled anti-human immunoglobulin antibody. After removing the unreacted labeled anti-human immunoglobulin antibody, the signal from the labeled anti-human immunoglobulin antibody bound to the carrier via the HBc antigen protein and the anti-HBc antibody is detected by an appropriate method in the sample. It becomes possible to specifically measure the contained anti-HBc antibody.

  In the secondary reaction step, if a labeled anti-human IgG antibody is used as a labeled anti-human immunoglobulin antibody, an IgG-type anti-HBc antibody can be measured, and if a labeled anti-human IgM antibody is used, an IgM-type anti-HBc antibody can be measured. Antibodies can be measured. When it is desired to measure both IgG anti-HBc antibody and IgM anti-HBc antibody, both labeled anti-human IgG antibody and labeled anti-human IgM antibody may be used. If it is not necessary to distinguish between the two, a labeled anti-human IgG antibody bound with the same labeling substance and a labeled anti-human IgM antibody may be mixed and subjected to a secondary reaction. If you want to distinguish between IgG-type anti-HBc antibody and IgM-type anti-HBc antibody, perform a secondary reaction using a labeled anti-human IgG antibody and labeled anti-human IgM antibody combined with different labeling substances at the same time, as described later. The detection process may be performed by distinguishing both, or the secondary reaction step may be performed separately. Further, as described later, an antigen-binding fragment may be used in place of the anti-human immunoglobulin antibody.

  Although the two-step method has been described above as an example, immunoassay may be performed by the one-step method. Alternatively, an anti-human immunoglobulin antibody can be bound to a solid support and measurement can be performed using a labeled HBc antigen protein. In this case, the reaction between the solid phase antibody and the diluted specimen is a primary reaction, and the reaction between the washed solid phase and the labeled HBc antigen is a secondary reaction. Further, as described above, the immunoassay method of the present invention is not limited to the sandwich method, and the anti-HBc antibody may be detected by a competition method.

  The HBc antigen used for measurement of the anti-HBc antibody may be any as long as it specifically binds to the anti-HBc antibody. Commercially available recombinant HBc antigens can be used, and recombinant HBc antigens can also be prepared and used by well-known genetic engineering techniques. It can also be obtained by extracting HBc antigen from hepatitis B virus-infected cells.

  The amino acid sequence of the HBc protein and the nucleotide sequence that encodes it can be obtained from databases such as NCBI's GenBank. Therefore, those skilled in the art can easily prepare recombinant HBc antigens using well-known genetic engineering techniques. can do. The sequences shown in SEQ ID Nos. 1 and 2 in the sequence listing are examples of known HBc genes and HBc protein sequences encoded thereby, and are registered in GenBank with accession No. AF324125.1. Most of the amino acid sequences of HBc proteins known so far have about 95% or more identity with the amino acid of SEQ ID NO: 2, or 1 to several amino acids are substituted, deleted, inserted and An HBc protein having an added amino acid sequence and having such an amino acid sequence can be preferably used as an HBc antigen for anti-HBc antibody measurement. However, the sequence of the HBc antigen is not limited to this, and the amino acid sequence of SEQ ID NO: 2 is less than 95% identical or an extra amino acid sequence such as a tag sequence is added to the N-terminal or C-terminal. Can be used as an HBc antigen as long as it has reactivity with an anti-HBc antibody induced in the body of an individual infected with HBV. In the present invention, “HBc antigen” includes the various HBc proteins described above.

  The amino acid sequence identity means that both amino acid sequences are aligned so that the amino acid residues of the two amino acid sequences to be compared match as much as possible, and the number of matching amino acid residues is divided by the total number of amino acid residues. The thing is expressed as a percentage. In the above alignment, a gap is appropriately inserted in one or both of the two sequences to be compared as necessary. Such sequence alignment can be performed using a known program such as BLAST, FASTA, CLUSTAL W, and the like. When gaps are inserted, the total number of amino acid residues is the number of residues obtained by counting one gap as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the identity (%) is the total number of amino acid residues in the longer sequence, and the number of amino acid residues matched. Is calculated by dividing. However, if the sequence to be compared is linked to any other sequence (for example, a tag sequence, a linker sequence, another protein sequence, etc.), such an arbitrary sequence is excluded and the identity is excluded. Is calculated.

  Twenty kinds of amino acids constituting natural proteins are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro) and neutral amino acids having hydrophilic side chains (Asn, Gln). , Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) Can be divided. It is known that substitution within these groups often does not change the properties of the protein. Therefore, even if the protein is partially different in amino acid sequence from the known HBc protein exemplified in SEQ ID NO: 2, it is possible that the binding to the anti-HBc antibody is maintained if the substitution is within these groups. high.

  The solid phase carrier is not particularly limited, and may be one conventionally used for immunoassay. For example, a plastic plate, fine particles, a fibrous substance, or the like can be used. Conventionally known materials can be used as the material of these carriers, and are not particularly limited. However, if the particles are fine particles, for example, various kinds of plastic beads such as glass beads and polystyrene, latex particles, and various types of ferrite particles (for example, JP-A-HEI 3-115862 publication). Examples of the fibrous substance include those containing cellulose, nitrocellulose, chitosan, polyethylene glycol polymer, silane polymer and the like as constituent components.

  For immobilization of the HBc antigen on these carriers, known methods such as physical adsorption and chemical bonding using a crosslinking agent can be used as appropriate.

The anti-immunoglobulin antibody used in the present invention may be any antibody that specifically binds to the class of anti-HBc antibody to be measured, such as an anti-IgG antibody or an antigen-binding fragment thereof, and an anti-IgM antibody or an antigen-binding property thereof. At least one of the fragments can be used. A suitable anti-immunoglobulin antibody derived from a specimen is preferably used. For example, when a specimen derived from a human is used, it is preferable to use an anti-human immunoglobulin antibody or an antigen-binding fragment thereof as described above. Specific examples of anti-immunoglobulin antibodies or antigen-binding fragments thereof include polyclonal antibodies obtained by immunizing laboratory animals such as mice, rabbits, chickens, goats, sheep and cows; spleen cells are isolated from the immunized experimental animals Monoclonal antibodies produced by hybridomas obtained by fusing with myeloma cells; Monoclonal antibodies produced by cells immortalized by EB virus from spleen cells or blood leukocytes isolated from immunized experimental animals; Recombinant antibodies; A molecule that exhibits high specificity and affinity for the anti-HBc antibody of the class to be measured, such as fragments of the above-mentioned antibodies having binding properties to globulin (eg, Fab, Fab ′, F (ab ′) ₂ , Fv, scFv). Anything can be used. As the anti-immunoglobulin antibody or antigen-binding fragment thereof, a commercially available product may be used, or an anti-immunoglobulin antibody or antigen-binding fragment thereof may be prepared and used by a known conventional method.

  Examples of the labeling substance used for labeling the anti-immunoglobulin antibody or antigen-binding fragment thereof or HBc antigen include enzymes, fluorescent substances, chemiluminescent substances, staining substances, radioactive substances and the like. As the enzyme, known ones such as alkaline phosphatase (ALP), peroxidase, β-galactosidase can be used, but the enzyme is not limited thereto. In order to provide a measurement system with high detection sensitivity, it is desirable to use ALP.

When an enzyme is used as a labeling substance, a chromogenic substrate, a fluorescent substrate or a luminescent substrate corresponding to the enzyme is added to the measurement system, and signals such as chromogenic or luminescent generated by the enzyme reaction are absorptiometers, luminometers, etc. What is necessary is just to measure using. For example, when ALP is used as a labeling substance, 3- (4-methoxyspiro (1,2-dioxetane-3,2′-tricyclo [3.3.1.1 ^3,7 ] decane) -4-yl) phenyl A luminescent substrate such as phosphate disodium (for example, trade name AMPPD (registered trademark)) can be used. When biotin or hapten is used as the labeling substance, streptavidin or a hapten antibody to which an enzyme, a fluorescent substance, a chemiluminescent substance, a staining substance, a radioactive substance or the like is bound may be used.

  The anti-immunoglobulin antibody may be labeled directly by a known covalent bond method or the like, or may be indirectly labeled using another binding pair such as biotin-avidin.

  When quantifying the anti-HBc antibody, the anti-HBc antibody is measured using the HBc antigen for a standard sample of known concentration containing the anti-HBc antibody at various concentrations, and the amount of the signal from the label and the amount in the standard sample are measured. A standard curve may be created by plotting the correlation with the anti-HBc antibody concentration. By performing the same operation on the target sample whose anti-HBc antibody concentration is unknown, measuring the amount of signal from the label, and applying the measured amount of signal to this standard curve, the anti-HBc antibody in the sample can be quantified. . Create a standard curve using WHO International Standard First International Standard for anti-Hepatitis B core antigen (anti-HBc), plasma, human. NIBSC code: 95/522 If the amount of anti-HBc antibody in the specimen derived from the test individual is calculated based on this standard curve, the amount of antibody can be expressed by the concentration (IU / mL) in international units. Alternatively, the amount of antibody can be determined at a concentration in international units (IU / mL) using another standard calibrated against the above international standard.

  When measuring an anti-HBc antibody, a negative control and a positive control may be measured in addition to the specimen. As a negative control, a blood sample confirmed to be negative for anti-HBc antibody, various buffers not containing anti-HBc antibody, and the like can be used. As a positive control, a solution obtained by adding the above-mentioned WHO first international standard for anti-HBc antibody at a predetermined concentration to a blood sample or buffer solution confirmed to be negative for anti-HBc antibody, or the blood sample Or a solution in which another standard calibrated against the above international standard is added to the buffer solution at a predetermined concentration, a solution in which an anti-HBc antibody positive specimen, or an anti-HBc antibody purified from humans is added at a predetermined concentration Etc. can be used.

  The present invention also provides an immunoassay kit for measuring an anti-HBc antibody in a specimen by the method of the present invention.

  In one embodiment, the kit comprises the above-described specimen diluent, that is, a solution containing at least one surfactant selected from (1) and (2) above, and an HBc antigen (Aspect A). . This aspect A includes aspects 1 and 3 of the aspects of the present invention described above, as will be described later. As described above, the specimen diluent preferably contains the surfactant at a concentration of 0.040 w / v% to 1.27 w / v% in the antigen-antibody reaction system. As described above, the surfactant concentration in the sample diluent is 0.25 w / v% to 8.02 w / v%, 0.063 w / v% to 2.01 w / v, depending on the amount of sample and sample diluent used. %, Values from 0.48 w / v% to 15.2 w / v% can be taken. The surfactant in the sample diluent is appropriately adjusted so that the surfactant concentration in the antigen-antibody reaction system (especially in the primary reaction system in the case of two steps) is 0.040 w / v% to 1.27 w / v%. Can be set.

  The immunoassay kit of the present invention may be an immunoassay kit by a sandwich method, and may further contain an anti-immunoglobulin antibody or an antigen-binding fragment thereof. Preferred conditions for the sample diluent, immunoassay method, HBc antigen, anti-immunoglobulin antibody, or antigen-binding fragment thereof are the same as described above. For example, the sample dilution liquid may further contain the above-described salt in addition to the predetermined surfactant. In addition, the anti-immunoglobulin antibody or antigen-binding fragment thereof may be an anti-IgG antibody or an anti-IgM antibody, and typically may include an anti-IgG antibody, particularly an anti-human IgG antibody.

  In the kit, the HBc antigen may be in a form bound to a solid phase carrier such as a plate or particle (eg, magnetic particle). In this case, the anti-immunoglobulin antibody or antigen-binding fragment thereof may be included in the kit in the form of an antibody solution dissolved in an appropriate medium, and typically, the anti-immunoglobulin antibody or antigen-binding fragment thereof. Can be included in the kit as a labeled antibody solution in a form labeled with a labeling substance. In this embodiment, the antibody solution may or may not contain the predetermined surfactant described above.

  When the HBc antigen is contained in a kit in the form of an antigen reagent solution contained in a liquid (for example, in the form of an antigen reagent solution containing an HBc antigen immobilized on a particle carrier), A surfactant may also be included in the antigen reagent solution. The kit containing the surfactant only in the sample diluent corresponds to the embodiment 1 of the above-described embodiments of the present invention, and the kit containing the surfactant in both the sample diluent and the antigen reagent solution is This corresponds to aspect 3 of the aspects of the present invention.

  For further reduction of non-specific reaction, the sample diluent may contain a salt, and instead of adding salt to the sample diluent, or in combination with the addition of salt to the sample diluent, the antigen reagent solution The salt concentration inside may be increased. That is, in the kit of the present invention, either one or both of the sample diluent and the antigen reagent solution may contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more.

  Alternatively, the HBc antigen may be included in the kit as a labeled antigen solution in a form labeled with a labeling substance. In this case, the anti-immunoglobulin antibody or antigen-binding fragment thereof can be included in the kit in a form bound to a solid phase carrier. In this embodiment, the labeled HBc antigen solution may or may not contain the predetermined surfactant described above.

  An antibody reagent solution containing an anti-immunoglobulin antibody or antigen-binding fragment thereof in a liquid form (for example, an antibody reagent solution containing an anti-immunoglobulin antibody or antigen-binding fragment thereof immobilized on a particle carrier) In the case of being included in the kit), the above-mentioned predetermined surfactant may be included in the antibody reagent solution. The kit containing the surfactant only in the sample diluent corresponds to the above-described embodiment 1 of the embodiments of the present invention, and the kit containing the surfactant in both the sample diluent and the antibody reagent solution is This corresponds to aspect 3 of the aspects of the present invention.

  In order to further reduce non-specific reactions, the sample diluent may contain a salt. Instead of adding salt to the sample diluent, or in combination with the addition of salt to the sample diluent, the antibody reagent solution The salt concentration inside may be increased. That is, in the kit of the present invention, either one or both of the sample diluent and the antibody reagent solution may contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more.

  In another embodiment, the immunoassay kit of the present invention includes an antigen reagent solution containing an HBc antigen, and the antigen reagent solution is at least one surfactant selected from (1) and (2) above. (Aspect B). This kit of aspect B corresponds to aspect 2 of the aspects of the present invention described above. The antigen reagent solution may be a reagent containing the antigen in a form in which the HBc antigen is immobilized on a particle carrier.

  In order to further reduce the non-specific reaction, the salt concentration in the antigen reagent solution may be increased, and the salt concentration in that case may be a concentration at which the salt concentration in the antigen-antibody reaction system is 158 mM or more. Alternatively, the kit may contain a sample diluent, and either or both of the sample diluent and the antigen reagent solution contain a salt such that the salt concentration in the antigen-antibody reaction system is 158 mM or more. You may do it.

  Other preferred conditions for the kit of aspect B are the same as in the immunoassay method according to aspect 2.

  In yet another embodiment, the immunoassay kit of the present invention comprises an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof, and an HBc antigen, wherein the antibody reagent solution comprises the above (1) and It contains at least one surfactant selected from (2) (Aspect C). This kit of embodiment C corresponds to embodiment 2 of the embodiments of the present invention described above. The antibody reagent solution can be a reagent containing the antibody or antigen-binding fragment thereof in a form in which an anti-immunoglobulin antibody or antigen-binding fragment thereof is immobilized on a particle carrier.

  In order to further reduce the non-specific reaction, the salt concentration in the antibody reagent solution may be increased, and the salt concentration in that case may be a concentration at which the salt concentration in the antigen-antibody reaction system is 158 mM or more. Alternatively, the kit may contain a sample diluent, and either or both of the sample diluent and the antibody reagent solution contain a salt so that the salt concentration in the antigen-antibody reaction system is 158 mM or more. You may do it.

  Other preferable conditions for the kit of aspect C are the same as those of the immunoassay method according to aspect 2.

  The anti-HBc antibody immunoassay kit of the present invention further includes other reagents generally contained in the immunoassay kit, such as an appropriate substrate solution and washing solution depending on the labeling substance to be used. May be.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Reference Example 1 Measurement method
(1) Production of HBc antigen-binding particles Recombinant HBc antigen-binding particles were prepared as follows.
Magnetic ferrite particles and recombinant HBc antigen were mixed at a weight ratio of 500: 1 in 0.1 M phosphate buffer and incubated at 25 ° C. for 1 hour with gentle stirring. After the reaction, ferrite particles are collected with a magnet and separated from the reaction solution. The particles are washed with 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2), and HBc antigen-binding particles. Got.

(2) Preparation of alkaline phosphatase-labeled anti-IgG antibody Mouse anti-human IgG monoclonal antibody and bovine small intestine-derived alkaline phosphatase (produced by Oriental Yeast Co., Ltd.) were used by the method of Yoshitake et al. , p1413-1424) to prepare an alkaline phosphatase-labeled antibody. As usual, desalted mouse anti-human IgG monoclonal antibody and pepsin were mixed in 0.1 M citrate buffer (pH 3.5) and left at 37 ° C. for 1 hour for pepsin digestion. After stopping the reaction, gel filtration purification was performed to obtain an anti-human IgG antibody from which the Fc region was removed. Next, 2-mercaptoethylamine hydrochloride was added to effect thiolation. Further, desalting treatment was performed to obtain an anti-human IgG antibody Fab.

On the other hand, desalted alkaline phosphatase and N- (4-maleimidobutyryloxy) -succinimide (GMBS) (final concentration 0.3 mg / mL) were mixed and allowed to stand at 30 ° C. for 1 hour for maleimidation. After desalting, Fab and maleimidated alkaline phosphatase were mixed at a molar ratio of 1: 1 and allowed to stand at 25 ° C. for 30 minutes for coupling. The coupling solution was allowed to stand at 15 ° C. for 1 hour, and 2-mercaptoethylamine hydrochloride was added to stop the reaction. After concentration and desalting, 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, 1 mM MgCl ₂ , 0.1 mM ZnCl ₂ , pH 7.2) was added to obtain an alkaline phosphatase-labeled anti-IgG antibody solution .

(3) Measurement of anti-HBc antibody 10 μL of the sample, negative control or positive control and 190 μL of the sample diluent were mixed.
As a sample, a negative serum sample (HBs antigen negative, HBcAb negative) purchased from ProMedDx was used. Samples 1 to 6 used in the following examples have been examined in detail and confirmed to be negative samples. The negative control and the positive control are the “standard negative solution for HBcAb-III” and “HBcAb-III” included in the “Lumipulse Presto (registered trademark) HBcAb-III” (Fujirebio Inc.) Standard positive solution "was used. As the sample dilution solution, a 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which each surfactant was added to a predetermined concentration was used.

  50 μL of HBc antigen-binding particles suspended in 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2) and 10 μL of diluted specimen were dispensed into a reaction vessel and stirred. Thereafter, incubation was performed at 37 ° C. for 8 minutes, and B / F separation and washing were performed. Thereafter, 50 μL of alkaline phosphatase-labeled anti-IgG antibody solution was dispensed into the reaction vessel, and after stirring, incubated at 37 ° C. for 8 minutes to separate and wash B / F. Then, Lumipulse substrate solution containing chemiluminescent substrate 3- (2'-spiroadamantane) -4-methoxy-4- (3 ''-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) 200 μL was dispensed into a reaction vessel, stirred and incubated at 37 ° C. for 4 minutes, and then the luminescence was measured with a luminometer to obtain the count number. The actual measurement was performed with a fully automatic chemiluminescent enzyme immunoassay system “Lumipulse Presto II” (Fujirebio Inc.). The cleaning solution and the Lumipulse substrate solution used were those attached to “Lumipulse Presto HBcAb-III”.

For the number of counts obtained in each measurement, from the ratio of the number of counts when using a sample diluent with a surfactant to the number of counts when using a sample diluent without adding a surfactant, The reduction rate of non-specific reaction was calculated and the effect of adding each surfactant was evaluated.
Nonspecific reaction reduction rate (%) = X / Y x 100
X: Count when using sample dilution with surfactant added
Y: Count when using sample dilution without surfactant

The following was set as a standard for evaluating that the addition of a surfactant was “effective”.
(1) Non-specific reaction reduction rate of positive control is 85% or more
(2) Reduction rate of non-specific reaction is less than 90% for 2 or more samples

Example 1. Addition of various concentrations of surfactant According to the measurement method described in Reference Example 1, anti-HBc antibodies in Samples 1 to 6 were measured. As the sample diluent, a diluent obtained by adding Triton X-405 (polyoxyethylene octylphenyl ether, HLB value 17.9), which is a nonionic surfactant, at each concentration was used.

  The results are shown in Table 1. Table 1 shows the number of counts and the nonspecific reaction reduction rate (%) in the case of using a sample dilution solution to which various concentrations of a surfactant were added. When a surfactant is added to the sample diluent at a concentration of 0.25% w / v or more so that the concentration in the primary reaction solution is 0.04 w / v% or more, the number of negative sample counts is reduced. Non-specific reaction reduction rate decreased to less than 90% in the sample. On the other hand, the count of the positive control hardly decreased, and the nonspecific reaction reduction rate was 85% or more. From these results, it has been clarified that non-specific reactions are suppressed by adding a surfactant to the sample diluent so that the concentration in the primary reaction solution is 0.04% or more.

Example 2 Addition of various nonionic surfactants In order to investigate other nonionic surfactants, anti-HBc antibodies were prepared in the same manner as in Example 1 using specimen diluents to which various nonionic surfactants were added. It was measured.

  The results are shown in Table 2. Table 2 shows the number of counts when using a sample diluent to which various surfactants were added at a concentration of 1.5 w / v% (the concentration of various surfactants in the primary reaction solution was 0.238 w / v%). The non-specific reaction reduction rate (%) is shown. Reduce non-specific reaction in multiple samples by adding nonionic surfactants of TritonX-100, TritonX-114, TritonX-305, TritonX-705, Brij58, BrijC10, Tween20, Tween40, Tween80 to the sample diluent The rate dropped to less than 90%. On the other hand, the nonspecific reaction reduction rate of the positive control was 85% or more. From these results, it was revealed that non-specific reaction is suppressed by adding a nonionic surfactant to the sample diluent. In particular, when Tritons (polyoxyethylene octylphenyl ether) was added, the nonspecific reaction was suppressed in many specimens, and the nonspecific reaction reduction rate was greatly reduced.

Example 3 Addition of Amphoteric Surfactant Further, in order to examine amphoteric surfactant, anti-HBc antibody was measured in the same manner as in Example 1 using a sample dilution solution to which amphoteric surfactant was added.

  The results are shown in Table 3. Table 3 shows the number of counts when various surfactants are added to the specimen diluent at a concentration of 1.5 w / v% (the concentration of various surfactants in the primary reaction solution is 0.238 w / v%). Specific reaction reduction rate (%) is shown. C12APS (N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C14APS (N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), C16APS (N- By adding hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate) to the sample diluent, the non-specific reaction reduction rate for a plurality of samples decreased to less than 90%. On the other hand, the reduction rate of non-specific reaction relative to the positive control was 85% or more. From these results, it became clear that non-specific reactions were suppressed by adding amphoteric surfactants to the sample diluent.

Example 4 Combination effect with salt concentration The combination of salt concentration and surfactant was investigated. The anti-HBc antibody was measured in the same manner as in Example 1 using a sample dilution solution in which salt was added to each concentration in 50 mM Tris buffer (1 mM EDTA · 2Na, 0.5% TritonX-405, pH 7.2). Sodium chloride was used as the salt. In addition, in order to determine the nonspecific reaction reduction rate as in Example 1, the anti-reaction rate was similarly used in the case of using a sample diluent (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which no surfactant was added. HBc antibody was measured.

  The results are shown in Table 4. When 200 mM, 500 mM, or 800 mM salt is added to the sample dilution (when the salt concentration in the primary reaction solution is 158 mM, 205 mM, or 253 mM), the nonspecific reaction reduction rate of the negative sample is 150 Compared with the salt concentration of mM, it was further reduced. From this result, it became clear that non-specific reaction can be further suppressed by adding salt to the specimen diluent so that the salt concentration in the primary reaction solution becomes 158 mM or more in addition to the surfactant.

Example 5 Combination Effect with Salt Concentration A combination of a high concentration salt and a surfactant was examined. As specimens, specimens 7 to 12 which were negative serum specimens (HBs antigen negative, HBcAb negative) purchased from ProMedDx were used. Samples 7 to 12 were examined in detail as in Reference Example 1 and confirmed to be negative samples. As a specimen dilution solution, a 50 mM Tris buffer solution (445 mM NaCl, 1 mM EDTA · 2Na, 0.24% TritonX-705, pH 7.2) containing 445 mM sodium chloride and 0.24% TritonX-705 was used. As HBc antigen-binding particles, 50 mM Tris buffer containing 445 mM sodium chloride and 0.24% TritonX-705 (445 mM NaCl, 0.1% sodium azide, 2% BSA, 0.24% TritonX-705, pH 7.2) Using the suspended HBc antigen-binding particles, anti-HBc antibody was measured in the same manner as in Example 1. Further, in order to obtain the nonspecific reaction reduction rate as in Example 1, 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) to which a surfactant was not added as a specimen dilution solution, Anti-HBc antibodies were similarly measured when HBc antigen-binding particles suspended in mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2) were used.

  The results are shown in Table 5. Negative when 445 mM sodium chloride and 0.24% TritonX-705 are added to the sample diluent and HBc antigen-binding particle suspension (when the salt concentration in the primary reaction solution is 443 mM and the TritonX-705 concentration is 0.238%) As a result, the non-specific reaction reduction rate was greatly reduced. The positive control maintained a high nonspecific reaction reduction rate, and no adverse effect on the positive count was observed. From this result, it was revealed that nonspecific reaction can be further suppressed when the salt concentration in the primary reaction solution is 443 mM in addition to the surfactant.

Example 6 Effect of addition of salt and surfactant when the amount of sample was increased The effect of addition of salt and surfactant when the amount of sample was increased was examined. The sample, negative control or positive control 10 μL and sample dilution liquid 190 μL were mixed. As samples, samples 13 to 18 which were negative serum samples (HBs antigen negative, HBcAb negative) purchased from ProMedDx were used. Samples 13 to 18 were examined in detail as in Reference Example 1 and confirmed to be negative samples. Use 50 mM Tris buffer solution (150 mM NaCl, 1 mM EDTA · 2Na, 0.25% TritonX-705, pH 7.2) containing 0.25% TritonX-705 as a sample diluent to examine the effect of adding surfactants. It was. Next, 50 μL of HBc antigen-binding particles suspended in 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, 0.25% TritonX-705, pH 7.2) containing 0.25% TritonX-705, 20 μL of diluted specimen was dispensed into a reaction vessel, and anti-HBc antibody was measured in the same manner as in Example 1. At this time, the concentration of TritonX-705 in the primary reaction solution is 0.246%. In addition, in order to examine the effects of addition of salt and surfactant, the specimen dilution solution was 50 mM Tris buffer containing 200 mM sodium chloride and 0.25% TritonX-705 (200 mM NaCl, 1 mM EDTA · 2Na, 0.25% TritonX- 705, pH 7.2), and as a suspension of HBc antigen-binding particles, 50 mM Tris buffer (200 mM NaCl, 0.1% sodium azide, 2% BSA, 0.25) containing 200 mM sodium chloride and 0.25% TritonX-705 Anti-HBc antibody was similarly measured using 50 μL of HBc antigen-binding particles suspended in% TritonX-705, pH 7.2). At this time, the salt concentration in the primary reaction solution is 199 mM, and the Triton X-705 concentration is 0.246%.

  The results are shown in Table 6. Even when the amount of the sample was increased, it was shown that by adding a surfactant during the primary reaction, the reduction rate of the non-specific reaction is reduced and the non-specific reaction can be suppressed. Furthermore, the nonspecific reaction reduction rate was significantly reduced by combining a high concentration salt and a surfactant. This indicates that the combination of the salt and the surfactant is effective in reducing the nonspecific reaction regardless of the amount of the sample.

Example 7 Effect of addition of surfactant to primary reaction solution The effect of addition of surfactant to the primary reaction solution was examined. As samples, samples 19 to 24, which were negative serum samples (HBs antigen negative, HBcAb negative) purchased from ProMedDx, were used. Samples 19 to 24 were examined in detail in the same manner as in Reference Example 1 and confirmed to be negative samples. As the sample dilution, 50 mM Tris buffer (150 mM NaCl, 1 mM EDTA · 2Na, pH 7.2) was used. As HBc antigen-binding particles, 50 mM Tris buffer (150 mM NaCl, 0.1% sodium azide, 2% BSA, pH 7.2, each containing 0.5% TritonX-305, TritonX-405 and TrironX-705) The anti-HBc antibody was measured in the same manner as in Example 1 using the HBc antigen-binding particles suspended in (1), and the nonspecific reaction reduction rate was determined.

  The results are shown in Table 7. When 0.5% of a surfactant was added to the HBc antigen-binding particle suspension (when the concentration of the surfactant in the primary reaction solution was 0.417%), the nonspecific reaction reduction rate was greatly reduced. From this result, it was confirmed that a non-specific reaction can be suppressed by including a surfactant in the primary reaction solution.

Claims (20)

An immunoassay method for anti-HBc antibody in a specimen, comprising the following (1) and (2):
(1) Nonionic surfactant
(2) An antigen-antibody reaction by contacting a specimen containing a reagent containing an HBc antigen or an anti-immunoglobulin antibody or an antigen-binding fragment thereof under conditions where at least one surfactant selected from amphoteric surfactants coexists. Performing the method.   The method according to claim 1, comprising mixing the specimen and the reagent containing at least one surfactant selected from (1) and (2) and performing the antigen-antibody reaction.   The method according to claim 1, comprising mixing the specimen, a solution containing at least one surfactant selected from (1) and (2) above, and the reagent to perform the antigen-antibody reaction. .   A reagent, a solution containing at least one surfactant selected from (1) and (2), and the reagent containing at least one surfactant selected from (1) and (2) And carrying out the antigen-antibody reaction.   A solution containing at least one surfactant selected from the above (1) and (2) and a sample are added to a reagent containing an HBc antigen or an anti-immunoglobulin antibody or an antigen-binding fragment thereof to carry out an antigen-antibody reaction. The method of claim 1 comprising performing.   The nonionic surfactant according to (1) is a surfactant selected from ether-type and ester ether-type nonionic surfactants having an HLB value of 10 to 20. The method according to claim 1.   The ether type nonionic surfactant is a surfactant selected from a polyoxyethylene alkylphenyl ether surfactant and a polyoxyethylene alkyl ether surfactant, and the ester ether type nonionic surfactant The method according to claim 6, wherein the surfactant is a polyoxyethylene sorbitan fatty acid ester surfactant.   The method according to any one of claims 1 to 7, wherein the amphoteric surfactant (2) is a sulfobetaine type surfactant.   The method according to any one of claims 1 to 8, wherein the concentration of the surfactant in the antigen-antibody reaction system is 0.040 w / v% to 1.27 w / v%.   The method according to claim 3, wherein the solution further comprises a salt.   The method according to any one of claims 1 to 10, wherein the reagent contains a salt such that a salt concentration in the reaction system of the antigen-antibody reaction is 158 mM or more.   The method according to claim 10 or 11, wherein the salt is at least one selected from sodium chloride and potassium chloride.   The method according to any one of claims 10 to 12, wherein the concentration of the salt in the antigen-antibody reaction system is 158 mM to 443 mM.   The method according to any one of claims 1 to 13, wherein the anti-HBc antibody comprises an IgG type antibody.   The method according to any one of claims 1 to 14, wherein the immunoassay is performed by a sandwich method.   The primary reaction is a reaction between the HBc antigen immobilized on the solid support and the specimen, and the secondary reaction is a solid support after B / F separation and washing. The method according to claim 15, which is a reaction between an anti-immunoglobulin antibody or an antigen-binding fragment thereof bound with a labeling substance.   The method according to claim 16, wherein the anti-immunoglobulin antibody or antigen-binding fragment thereof bound with a labeling substance comprises an anti-human IgG antibody or antigen-binding fragment thereof bound with a labeling substance. An anti-HBc antibody immunoassay kit comprising the following (1) and (2):
(1) Nonionic surfactant
(2) A kit comprising a solution containing at least one surfactant selected from amphoteric surfactants and an HBc antigen. An anti-HBc antibody immunoassay kit comprising an antigen reagent solution containing an HBc antigen, the reagent solution comprising the following (1) and (2):
(1) Nonionic surfactant
(2) A kit containing at least one surfactant selected from amphoteric surfactants. An anti-HBc antibody immunoassay kit comprising an antibody reagent solution containing an anti-immunoglobulin antibody or an antigen-binding fragment thereof and an HBc antigen, wherein the antibody reagent solution comprises the following (1) and (2):
(1) Nonionic surfactant
(2) A kit containing at least one surfactant selected from amphoteric surfactants.