JP2017067513A - Method of measuring human parvovirus b19 antigen - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means of further mitigating reduction in measurements caused by host-derived antigens in a sample in human parvovirus B19 antigen immunoassay.SOLUTION: Provided is a human parvovirus B19 antigen immunoassay method which includes a step of treating a sample under an acidic environment with pH of 3.5 or less using a treatment liquid containing at least one of the following surfactants (1)-(4): (1) an ampholytic surfactant having an alkyl group with a carbon number of 10 or greater and quaternary ammonium in a molecule, (2) a cationic surfactant having an alkyl group with a carbon number of 10 or greater and quaternary ammonium in a molecule, (3) a non-ionic surfactant having an alkyl group with a carbon number of 9 or greater and tertiary amide in a molecule, and (4) an ampholytic surfactant having a steroid skeleton and quaternary ammonium in a molecule.SELECTED DRAWING: None

Description

  The present invention relates to an immunoassay method for human parvovirus B19 antigen.

  Human parvovirus B19 is a single-stranded DNA virus belonging to the genus Parvovirus, Parvoviridae. The virus particle has an icosahedral structure, and the capsid proteins VP-1 (83 kDa) and VP-2 (58 kDa) constitute a capsid at a ratio of 1: 9. It does not have an envelope. Known diseases / symptoms caused by parvovirus B19 infection include infectious erythema (apple disease), arthritis, fetal edema and miscarriage.

  As a method for easily and sensitively measuring human parvovirus B19 antigen in a sample, a method of performing immunoassay with an anti-human parvovirus B19 antibody after treating the sample under acidic conditions of pH 3.5 or lower is known (Patent Document 1). In addition, a technique is known in which an immunoassay is performed after treating a specimen with guanidine under acidic conditions having a slightly higher pH (Patent Document 2).

  In the method of measuring pathogen-derived antigens in specimens by immunological techniques, when antibodies against antigenic substances such as neutralizing antibodies are generated in the infected body, the host-derived antibodies present in the specimens measure the antigens. The antibody may compete with the antibody to form a complex with the host-derived antibody, and as a result, the measured value may decrease. This can be a particularly serious problem when trying to measure an antigen that is contained only in a trace amount in a specimen. The methods of Patent Document 1 and Patent Document 2 can improve the decrease in the measured value due to complex formation between the human parvovirus B19 antigen and the host-derived antibody to some extent, but there is still room for improvement.

  In Patent Document 3, in the immunoassay of hepatitis C virus (HCV) antigen in a specimen, the amount of virus in the body is obtained by pretreating the specimen with an acidifying agent and a cationic surfactant having a specific structure. A technique for increasing the detection sensitivity of HCV virus antigens with low HCV is disclosed. In this sample processing method, a host surfactant antibody in the sample is deactivated by treatment with an acidifying agent, and a cationic surfactant having a specific structure is added for the purpose of suppressing precipitation and white turbidity caused by acid treatment. HCV is a virus having an envelope. As described in Patent Document 2, in the case of a virus having an envelope, in order to detect a virus antigen, the envelope membrane is strongly destroyed by pretreatment, and the antigen in the membrane is detected. Protein needs to be exposed. Patent Document 3 clearly states that the target virus is a virus having a lipid membrane (that is, an envelope), although there is only a part of the description that the treatment method is applicable to human parvovirus. In the examples, only detection of HCV is described.

Patent No. 4449171 Japanese Patent No.4855126 Patent No. 4744298

  As described above, in the immunoassay of a virus antigen protein such as human parvovirus B19 that does not have an envelope, it is required to further improve the decrease in the measured value due to the host-derived antibody in the sample. The object of the present invention is to provide means for solving this problem in the immunoassay of human parvovirus B19.

  As a result of earnest research for the purpose of further improving the sensitivity of the immunoassay method for human parvovirus B19 antigen, the inventors of the present application have treated the specimen with a specific surfactant under acidic conditions of pH 3.5 or lower and As a result of the above reaction, it was found that the decrease in the measured value due to competition between the antibody used for immunoassay and the host-derived antibody can be suppressed, and the detection sensitivity can be further increased, and the present invention has been completed.

That is, the present invention includes a human parvovirus B19 antigen comprising treating a specimen under acidic conditions of pH 3.5 or lower with a treatment liquid containing at least one of the following surfactants (1) to (4): An immunoassay method is provided.
(1) Amphoteric surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(3) Nonionic surfactant having an alkyl group having 9 or more carbon atoms and a tertiary amide in the molecule
(4) Amphoteric surfactant having steroid skeleton and quaternary ammonium in the molecule

  Furthermore, the present invention provides a human parvovirus B19 antigen comprising a sample treatment solution containing at least one of the surfactants (1) to (4) above, and an anti-human parvovirus B19 antibody or an antigen-binding fragment thereof. An immunoassay kit is provided.

  According to the present invention, in the immunoassay of the antigen protein of human parvovirus B19 having no envelope, even if a host-derived antibody such as a neutralizing antibody that competes with the antibody used for detection is present in the specimen, The complex formed between the derived antibody and the antigen to be measured can be dissociated to prevent a decrease in the measured value. According to the present invention, the detection sensitivity can be further increased as compared with the conventional immunoassay for parvovirus B19 antigen.

In the present invention, a specimen to be measured for human parvovirus B19 antigen is treated with a treatment solution containing a specific surfactant under acidic conditions of pH 3.5 or lower. The surfactant used is at least one of the following (1) to (4).
(1) Amphoteric surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(3) Nonionic surfactant having an alkyl group having 9 or more carbon atoms and a tertiary amide in the molecule
(4) Amphoteric surfactant having steroid skeleton and quaternary ammonium in the molecule

In the amphoteric surfactant (1), the alkyl group is preferably a linear alkyl group. More preferably, the alkyl group has 12 or more carbon atoms. (1) Preferred examples of the surfactant, CH ₃ - In (n is 9 or more ^{_{integer) (CH 2) n -N +}} (CH 3) 2 - - [(CH 2) 3 -SO 3] Examples of the amphoteric surfactant having the structure shown can include surfactants shown in Table 1 below, but are not limited thereto.

In the cationic surfactant (2), the alkyl group is preferably a linear alkyl group. More preferably, the alkyl group has 12 or more carbon atoms. Preferred examples of surfactants _{(2), CH 3 - (} CH 2) n -N + (CH 3) 3 · Br -, and _{CH 3 - (CH 2) n} -N + (CH 3) 3 · Cl ^- (either n is 9 or more integer) can be mentioned cationic surfactant represented by, there may be mentioned a surfactant shown in table 2 as a specific example, limited to Not.

  In the nonionic surfactant (3), the alkyl group is preferably a linear alkyl group. Preferable examples of the surfactant of (3) include MEGA-10 (n-Decanoyl-N-methylglucamide) and MEGA-12 (n-Dodecanoyl-N-methylglucamide). MEGA-10 is more preferred. MEGA-10 is a nonionic surfactant having the following structure. MEGA-10 contains a straight-chain alkyl group having 9 carbon atoms and a tertiary amide in the molecule, and has a structure similar to the preferred embodiments of (1) and (2) described above. MEGA-12 also contains a straight chain alkyl group having 11 carbon atoms and a tertiary amide in the molecule, and has a structure similar to the preferred specific examples of (1) and (2) described above.

Preferred examples of amphoteric surfactants in (4) include CHAPS (3-[(3-Cholamidopropyl) dimethylammonio] -1-propanesulfonate) and CHAPSO (3-[(3-Cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonate). Can be mentioned. CHAPS is more preferred. CHAPS is an amphoteric surfactant having the following structure, -N ⁺ (CH ₃₎ comprising a quaternary ammonium in the molecule _{2 - [(CH 2) 3} -SO 3 -] amphoteric surfactant having the structure In that respect, it has a feature common to the above-described preferred specific example (1). CHAPSO also, -N ⁺ (CH ₃₎ comprising a quaternary ammonium in the molecule _{2 - [CH 2 -CHOH-CH} 2 -SO 3 -] structure in that it is an amphoteric surfactant having the above-mentioned ( It has features very similar to the preferred embodiment of 1).

  Among the surfactants of (1) to (4), surfactants that can be preferably used include C12APS, C14APS, C16APS, C12TAB, C14TAB, C12TAC, and C14TAC. In the present invention, C12APS is particularly preferred. It can be particularly preferably used.

  The concentration of the surfactants (1) to (4) in the treatment liquid may be about 0.01% to 5.0%, for example, about 0.01% to 3.0%, or about 0.05% to 2.5%. When a plurality of surfactants (1) to (4) are used in combination, each surfactant may have the above-mentioned concentration, but the total is preferably about 10% or less.

  In the present invention, in addition to the treatment with the surfactants (1) to (4) described above, a polyoxyethylene alkylphenyl ether-based nonionic surfactant and a polysorbate-based nonionic surfactant are also used. You may perform the sample process by the at least 1 type selected. The sensitivity of immunoassay can be further enhanced by using any one of the surfactants (1) to (4) in combination with these nonionic surfactants. The concentration of these nonionic surfactants that can be used in combination in the treatment liquid may be the same as that of the surfactants (1) to (4). If these nonionic surfactants are further added to the treatment liquid, the work process is simple. However, the treatment with the above treatment liquid is performed after mixing these nonionic surfactants and the sample first. These nonionic surfactants may be added after the treatment or treatment with the treatment liquid.

  Examples of the polyoxyethylene alkylphenyl ether surfactant include nonionic surfactants such as Triton X-100, Triton X-305, Triton X-405, Triton X-705, and Nonidet P-40. The polyoxyethylene alkylphenyl ether surfactant is also called a Triton surfactant.

  Polysorbate surfactants include polysorbate 20 (polyoxyethylene sorbitan monolaurate, trade name Tween 20), polysorbate 40 (polyoxyethylene sorbitan monopalmitate, trade name Tween 40), polysorbate 60 (polyoxyethylene sorbitan monostearate). , Trade name Tween60), polysorbate 65 (polyoxyethylene sorbitan tristearate, trade name Tween65), polysorbate 80 (polyoxyethylene sorbitan monooleate, trade name Tween80), polysorbate 85 (polyoxyethylene sorbitan trioleate, trade name) Nonionic surfactants such as Tween 85) are included. The polysorbate surfactant is also called a Tween surfactant or a polyoxyethylene sorbitan surfactant.

  Specific preferred examples of the polyoxyethylene alkylphenyl ether surfactant and polysorbate surfactant include Triton X-705, polysorbate 20, and polysorbate 80, but are not limited thereto.

  Alternatively, in addition to the treatment with the surfactants (1) to (4) described above, a sample treatment with a denaturing agent may be further performed. Specific examples of the modifying agent include guanidine, guanidine salts, guanidine derivatives, and urea. The sensitivity of immunoassay can be further increased when the surfactants of any one of (1) to (4) and a denaturant are used in combination. Specific examples of the guanidine salt include, but are not limited to, guanidine hydrochloride, guanidine carbonate, guanidine nitrate, guanidine phosphate, and guanidine sulfamate. Specific examples of the guanidine derivative include, but are not limited to, guanidinobenzoic acid, guanidinoglutaric acid, guanidinosuccinic acid, guanidinoacetic acid, guanidinopropionic acid, guanidinobenzimidazole, and the like. The concentration of the denaturing agent in the processing solution may be about 0.5M to 5M. A denaturing agent may be further added to the treatment liquid, or the denaturing agent treatment may be performed before or after the treatment with the treatment liquid.

  In the present invention, treating a specimen under acidic conditions of pH 3.5 or less means that the specimen solution being processed (that is, a solution in which the specimen and the treatment liquid are mixed) has a pH of 3.5 or less. The pH of the sample solution being processed may be, for example, about pH 3.0 or lower, or about pH 2.8 or lower. There are no particular limitations on the acid that can be used to bring the sample solution being processed into such acidic conditions, and both inorganic and organic acids can be used. For example, acids such as hydrochloric acid, sulfuric acid, acetic acid and citric acid can be used alone or in combination. Further, in order to make the sample solution being processed into an acidic condition, for example, an acidic buffer solution such as a glycine hydrochloride buffer solution or a citrate buffer solution adjusted to an appropriate pH may be used. An appropriate amount of acid or acidic buffer solution may be added to the processing solution, or an acid or acidic buffer solution may be mixed with the specimen separately from the processing solution.

  Other components are not particularly limited as long as the treatment liquid contains the above-described surfactant at a predetermined concentration. Add the above surfactant to the sample diluent used in commercially available immunoassay kits, etc., and add acid or acidic buffer so that the pH of the sample solution being processed is in the acidic condition as described above. Can be prepared. Alternatively, the acid or acidic buffer solution may be added to the specimen separately from the treatment solution as described above.

  The sample is treated with the treatment solution before the reaction with the antibody. The temperature at the time of sample treatment may be 0 ° C. to 50 ° C., and treatment at around room temperature is usually preferable. The treatment time is usually about 10 seconds to 10 minutes, but the treatment time may be appropriately adjusted according to the pH value and the treatment temperature.

  After the sample is treated by mixing the treatment liquid and the sample, the sample solution is neutralized with an alkaline solution to adjust the pH to near neutrality. Neutralization may be performed before the reaction with the antibody, or may be performed simultaneously with the reaction with the antibody. For example, by increasing the pH of the antibody solution (for example, about pH 8.5 to pH 10.0), neutralization can be performed simultaneously with the antibody reaction by mixing the acidified treated specimen with the antibody solution. it can.

The anti-human parvovirus B19 antibody used for immunoassay of human parvovirus B19 antigen in the treated specimen may be a polyclonal antibody or a monoclonal antibody. In general, monoclonal antibodies are preferably used. Further, antigen-binding fragments such as Fab ′, F (ab ′) ₂ and scFv can be used instead of antibodies. Anti-human parvovirus B19 antibodies are known and are also used in commercial kits. Such known antibodies can be preferably used. Alternatively, since methods for producing polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments are well known, anti-human parvovirus B19 antibodies or antigen-binding fragments thereof are prepared using human parvovirus B19 antigen as an immunogen, and immunoassay is performed. May be used for The human parvovirus B19 antigen protein used as an immunogen can be prepared, for example, with reference to JP-A-77-147986. Alternatively, it can be obtained by extraction and purification from human serum infected with human parvovirus.

  As used herein, the term “human parvovirus B19 antigen” includes the capsid proteins VP-1 and VP-2. The anti-human parvovirus B19 antibody for detecting the human parvovirus B19 antigen is not particularly limited, but it is preferable to use an anti-VP-2 antibody against VP-2, which has a high proportion of virus particles.

  The immunoassay of human parvovirus B19 antigen in the present invention can be carried out by a known immunoassay method such as a competitive method or a sandwich method. Among them, the sandwich method can be preferably employed in the present invention. Specific examples of sandwich immunoassay include chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, electrochemiluminescence immunoassay, etc. There are various methods. Any method may be used in the present invention.

  In a sandwich measurement system, usually, one of two types of antibodies or antigen-binding fragments thereof is used as a solid phase antibody immobilized on a solid phase, and the other is used as a labeled antibody labeled with a labeling substance. In the processed specimen treated according to the present invention, it is considered that parvovirus B19 antigen is mostly present as a monomer, so the solid phase antibody and labeled antibody used in the sandwich method recognize different sites of B19 antigen. It is desirable to use one.

  The solid phase is not particularly limited, and may be the same as the solid phase used in a known sandwich immunoassay system. Specific examples of the solid phase material include, but are not limited to, polystyrene, polyethylene, sepharose, and the like. The physical form of the solid phase is not critical in nature. The solid phase to be used is preferably one in which the antibody can be easily immobilized on the surface and the immune complex formed during the measurement and unreacted components can be easily separated. In particular, plastic plates and magnetic particles used in ordinary immunoassays are preferred. From the viewpoints of handling, storage, and ease of separation, it is most preferable to use magnetic particles made of the materials described above. The antibody can be bound to these solid phases by a conventional method well known to those skilled in the art.

  The labeling substance is not particularly limited, and the same labeling substance used in known immunoassay systems can be used. Specific examples include enzymes, fluorescent materials, chemiluminescent materials, staining materials, radioactive materials, 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.

  Briefly describing the procedure of sandwich immunoassay, first, a treated specimen is reacted with a solid phase antibody, washed and B / F separated, and then reacted with a labeled antibody. After washing again and B / F separation, signals from the labeled antibody (fluorescence, luminescence, color development, etc.) are detected. When an enzyme is used as the labeling substance, an appropriate substrate may be added and reacted to detect a signal generated by the enzyme reaction. Perform an immunoassay on a standard sample of known concentration containing human parvovirus B19 antigen at various concentrations, and create a calibration curve by plotting the correlation between the amount of signal from the label and the concentration of the anti-source in the standard sample. If so, the amount of the human parvovirus B19 antigen in the sample can be quantitatively measured.

  Although the above is a description of the procedure by the two-step method, a one-step method in which the treated specimen, the solid phase antibody and the labeled antibody are reacted at the same time may be used.

  The specimen subjected to the method of the present invention is typically a specimen isolated from a human. If the sample provider is infected with human parvovirus B19, it is not particularly limited as long as the sample is expected to contain the antigen of the virus. For example, serum, plasma, blood-derived sample such as blood for transfusion, Examples thereof include plasma fractionated preparations.

  An immunoassay kit for carrying out the immunoassay of the present invention comprises a sample treatment solution containing at least any one of the surfactants (1) to (4), an anti-human parvovirus B19 antibody or an antigen binding property thereof. Including fragments. The sample treatment solution of the kit may contain an acid or an acidic buffer so that the pH of the sample solution being treated is in the acidic condition as described above, or an acid solution such as an acidic buffer may be treated with the sample. It may be contained separately from the liquid. In addition, the kit includes at least one selected from a denaturant as described above, a polyoxyethylene alkylphenyl ether nonionic surfactant, and a polysorbate nonionic surfactant in the sample treatment solution. Further, it may be included, or may be included separately from the sample processing solution and added as appropriate during sample processing.

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

<Preparation of anti-parvovirus antibody-immobilized particles>
Mouse anti-parvovirus B19 monoclonal antibody Prv334 antibody 0.2 mg / mL was added to magnetic particles 0.01 g / mL in 10 mM MES buffer (pH 5.0), and incubated at 25 ° C. for 1 hour with gentle stirring. After the reaction, the magnetic particles were collected with a magnet, and the particles were washed with a washing solution (50 mM Tris buffer, 150 mM NaCl, 2.0% BSA, pH 7.2) to obtain anti-parvovirus antibody-immobilized particles. At the time of measurement, anti-parvovirus antibody-immobilized particles were suspended in a particle diluent (400 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN ₃ , 2.0% BSA, pH 9.0).

<Preparation of antibody labeled with alkaline phosphatase>
Demineralized alkaline phosphatase (ALP) 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. Subsequently, in a reaction solution for coupling (100 mM phosphate buffer, 1 mM EDTA2Na, pH 6.3), Fab′-modified mouse anti-parvovirus B19 monoclonal antibody VP2-312 antibody and maleimidated ALP were mixed at a molar ratio of 1: 1. The mixture was mixed at a ratio and reacted at 25 ° C for 1 hour. Using column chromatography on Superdex200 10/300 (GE), purification buffer (100 mM Tris buffer, 150 mM NaCl, 0.1% NaN ₃ , pH 8.0) with a flow rate of 0.5 mL / min. The peak was separated and purified to obtain an alkaline phosphatase-labeled antibody. At the time of measurement, alkaline phosphatase-labeled antibody was suspended in a labeled diluent (50 mM Tris buffer, 100 mM NaCl, 0.3 mM ZnCl ₂ , 1 mM MgCl ₂ , 0.1% NaN ₃ , 2.0% BSA, pH 7.2).

<Measurement of parvovirus B19 antigen>
20 μL of the pretreatment solution and 100 μL of the sample were dispensed into a reaction vessel, stirred and incubated at 37 ° C. for 6.5 minutes. Thereafter, 50 μL of antibody-immobilized particles were dispensed and stirred. Then, it incubated at 37 degreeC for 8 minute (s), and B / F separation and washing were performed. 50 μL of enzyme-labeled antibody 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 the reaction vessel, and after stirring, incubated at 37 ° C. for 4 minutes, the amount of luminescence was measured with a luminometer. The actual measurement was performed with a fully automatic chemiluminescent enzyme immunoassay system (Lumipulse Presto II (Fujirebio)).

1. Comparison of effects of various surfactants Various surfactants shown in Table 5 below were added to the pretreatment liquid, and the sample was pretreated. The pH of the pretreatment solution was appropriately adjusted with a glycine hydrochloride buffer so that the sample solution during the pretreatment had a pH of 2.8. As a sample, a purified human inactivated antigen dissolved in Tris buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA2Na, 0.1% NaN ₃ , 2.0% BSA, pH 7.2) was used as a positive sample. Moreover, what added the inhibitory antibody to the positive sample was used as a model of the specimen containing the antibody used for the reagent and the antibody to be batted. Two types of monoclonal antibodies (antibody 1 and antibody 2) capable of competing with the solid phase antibody Prv334 for binding to the parvovirus B19 antigen were used as inhibitory antibodies. From the number of counts in Lumipulse Presto II, the bond separation rate was calculated by the following formula, and the effect of each surfactant was evaluated.

  Binding separation rate (%) = (count value of positive sample to which inhibitory antibody was added) / (count value of positive sample to which inhibitory antibody was not added) × 100

  The results are shown in Table 5. When the sample was treated with an acidic pretreatment solution to which C12TAB, C12APS, C14APS, or C16APS was added, the antibody binding separation rate was greatly improved.

2. Examination of influence of pH during pretreatment The influence of pH during pretreatment on the effect of surfactant was evaluated. C12APS was used as a surfactant. The concentration of the surfactant in the pretreatment liquid was 2%. As samples, a negative sample (NC) (Tris buffer), a positive sample (PC), and a sample obtained by adding an inhibitory antibody to a positive sample were used. In addition to antibody 1 and antibody 2 described above, a monoclonal antibody (antibody 3) capable of competing with labeled antibody VP2-312 was used as an inhibitory antibody.

  The results are shown in Table 6. Table 6 shows the count value measured under each addition condition, the binding separation rate calculated from the count value, and the ratio (%) of the binding separation rate under each addition condition to the binding separation rate under the non-addition condition. Under all the conditions of pH 1.9 to 3.5 examined, the binding separation rate was improved by the addition of C12APS. As the pH was lowered, the bond separation rate tended to improve more.

3. Examination of alkyl chain length of surfactant The effect of the alkyl chain length of the surfactant on the bond separation rate was evaluated. The surfactants shown in Table 7 below were examined. The surfactant concentration in the treatment liquid was 2%, and the pH during the treatment was 2.4. The sample is 2. The same was used.

  The results are shown in Table 7. A surfactant having an alkyl chain with 10 or more carbon atoms showed an effect of improving the bond separation rate. Moreover, it turned out that a cationic surfactant shows an effect similarly.

4). Confirmation of the effect of the surfactant in the actual sample The effect of the surfactant was evaluated using a human serum sample. As a serum sample, 3321-146-2 (BBI2, 1.71 × 10 ¹¹ IU / mL) purchased from BBI was used. A sample obtained by diluting this specimen 10 ⁿ (n = 3 to 8) times with a diluted solution for Lumipulse was measured. The inhibitory antibody was used by mixing antibodies 1 to 3 and added so that the final concentration of the mixed antibody was 100 μg / mL. This antibody concentration is not much different from the amount of specific IgG produced in human blood. As the surfactant, C12APS was used and added to the pretreatment liquid so as to be 2%. The pH of the sample solution during the pretreatment was adjusted to 2.4. A plurality of negative samples and positive samples were measured in advance under the condition that neither the surfactant nor the inhibitory antibody was added, and the cut-off value was calculated from the obtained count values. In this measurement system, a positive sample can be determined if the count value is 5500 or more.

  The results are shown in Table 8. The DNA titer in the table is the parvovirus DNA titer (IU / mL). When inhibitor antibody is added, sensitivity decreases about 100 times when no surfactant is added, but by adding C12APS, the sensitivity is the same as when no inhibitor antibody is added even in the presence of inhibitor antibody. Had. That is, in the presence of an inhibitory antibody, the sensitivity increased 100-fold by adding a specific surfactant. As a result, it was confirmed that the effect of the present technology can be obtained even with an actual human serum sample.

5. Examination of effects of MEGA8, MEGA10 and CHAPS The effects of amphoteric surfactants CHAPS and nonionic surfactants MEGA8 and MEGA10 were evaluated. The surfactant was added to the pretreatment liquid so as to have a concentration of 2%. The pH during the pretreatment was 2.4. The sample is 2. The same was used.

  The results are shown in Table 9. For nonionic surfactants, MEGA10 significantly improved the bond separation rate. With CHAPS (a zwitterionic surfactant), a remarkable effect was observed when antibody 3 was added in which the binding site to parvovirus B19 was batted with the labeled antibody.

6). Examination of effect of C12APS + nonionic surfactant
The effect of using a nonionic surfactant in combination with C12APS was evaluated. The C12APS concentration in the treatment solution was 2%, and a nonionic surfactant was further added to the concentration to 2%. The pH during the pretreatment was 2.4. The sample is 2. In addition to the same 5 types, those in which each inhibitory antibody was added to the positive sample in a 10-fold amount (antibody 1 × 10, antibody 2 × 10, antibody 3 × 10) were used.

  The results are shown in Table 10. When C12APS was used in combination with a nonionic surfactant, the bond separation rate was further improved.

7). Examination of effect of C12APS + modifier
The effect of using C12APS in combination with a modifier was evaluated. Urea (2M, 4M) and guanidine (4M) were used as denaturing agents. The pH during pretreatment was 2.8. The sample is 6. The same was used.

  The results are shown in Table 11. When C12APS was used in combination with a modifier, the bond separation rate was further improved.

8). Examination of surfactant concentration The concentration of the surfactant added to the pretreatment solution was examined. C12APS was used as the surfactant, and the addition concentration in the pretreatment liquid was 0.1% to 2.0% as shown in Table 12 below. The pH during the pretreatment was 2.4. The sample is 2. The same was used.

  The results are shown in Table 12. The bond separation rate was improved under all conditions from 0.1% to 2.0%. If the surfactant is present in the pretreatment liquid at a concentration of 0.1% or more, an improvement effect is considered to be obtained. It was confirmed that the bond separation rate increased depending on the surfactant concentration.

9. (Comparative example) Examination of the effect of urea The effect of using urea alone instead of the surfactant under acidic conditions was confirmed. Urea was added at a concentration shown in Table 13 below to the pretreatment liquid adjusted so that the pH during pretreatment was 2.4. The sample is 2. The same was used.

  The results are shown in Table 13. When urea was used at a concentration of 4M or higher, the count value decreased significantly even for positive samples. Addition of 2M urea maintaining the count value did not show an effect of improving the binding separation rate.

Claims (11)

A method for immunoassay of human parvovirus B19 antigen, comprising treating a specimen under acidic conditions of pH 3.5 or lower with a treatment liquid containing at least one of the following surfactants (1) to (4).
(1) Amphoteric surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(3) Nonionic surfactant having an alkyl group having 9 or more carbon atoms and a tertiary amide in the molecule
(4) Amphoteric surfactant having steroid skeleton and quaternary ammonium in the molecule   The method according to claim 1, wherein the alkyl group in (1) to (3) is a linear alkyl group.   The method according to claim 1 or 2, wherein the amphoteric surfactant in (1) is a surfactant selected from C12APS, C14APS, and C16APS.   The method according to any one of claims 1 to 3, wherein the cationic surfactant (2) is a surfactant selected from C12TAB, C14TAB, C16TAB, C12TAC, C14TAC, and C16TAC.   The method according to any one of claims 1 to 4, wherein the nonionic surfactant (3) is MEGA-10 and the amphoteric surfactant (4) is CHAPS.   The treatment according to any one of claims 1 to 5, further comprising treating the specimen with at least one nonionic surfactant selected from a polyoxyethylene alkylphenyl ether surfactant and a polysorbate surfactant. The method described.   The method according to claim 6, wherein the at least one nonionic surfactant is at least one selected from Triton X-705, polysorbate 20, and polysorbate 80.   The method according to claim 1, further comprising treating the specimen with a denaturing agent.   The method according to claim 8, wherein the denaturing agent is at least one selected from guanidine, guanidine salts, guanidine derivatives, and urea.   The method according to any one of claims 1 to 9, wherein the immunoassay is performed by a sandwich method. A human parvovirus B19 antigen immunoassay kit comprising a sample treatment solution containing at least one of the following surfactants (1) to (4) and an anti-human parvovirus B19 antibody or an antigen-binding fragment thereof.
(1) Amphoteric surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and quaternary ammonium in the molecule
(3) Nonionic surfactant having an alkyl group having 9 or more carbon atoms and a tertiary amide in the molecule
(4) Amphoteric surfactant having steroid skeleton and quaternary ammonium in the molecule