JP6641841B2 - Method for measuring human parvovirus B19 antigen - Google Patents

Description

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

  Human parvovirus B19 is a single-stranded DNA virus belonging to the parvovirus family, parvovirus genus. The virus particle has an icosahedral structure, and the capsid proteins VP-1 (83 kDa) and VP-2 (58 kDa) constitute a capsid in a ratio of 1: 9. There is no envelope. Known erythema infectious disease (apple disease), arthritis, hydrops fetalis, and miscarriage are known as diseases and symptoms caused by parvovirus B19 infection.

  As a simple and sensitive method for measuring human parvovirus B19 antigen in a sample, there is known a method of treating a sample under acidic conditions of pH 3.5 or less and then performing an immunoassay with an anti-human parvovirus B19 antibody. (Patent Document 1). In addition, a method is also known in which an immunoassay is performed after treating a sample with guanidine under acidic conditions having a slightly higher pH (Patent Document 2).

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

  Patent Document 3 discloses that in an immunoassay for hepatitis C virus (HCV) antigen in a sample, the amount of virus in the body is determined by pretreating the sample with an acidifying agent and a cationic surfactant having a specific structure. A technique has been disclosed for increasing the detection sensitivity of HCV virus antigens with a small amount of HCV. In this sample processing method, a cationic surfactant having a specific structure is added for the purpose of inactivating host-derived antibodies in the sample by treatment with an acidifying agent and suppressing precipitation or cloudiness caused by the 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 removed. The protein needs to be exposed. Patent Literature 3 describes that the treatment method is applicable to a human parvovirus in only a few cases, but specifies that the target virus is a virus having a lipid membrane (that is, an envelope). In the examples, only the detection of HCV is described.

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

  As described above, in immunoassay of an antigen protein of a virus having no envelope such as human parvovirus B19, it is required to further improve the decrease in the measurement value due to the host-derived antibody in the sample. An object of the present invention is to provide a means capable of solving this problem in an immunoassay for human parvovirus B19.

  The present inventors have conducted intensive studies with the aim of further improving the sensitivity of the immunoassay method for the human parvovirus B19 antigen.As a result, the antibody was treated with a specific surfactant under acidic conditions at a pH of 3.5 or less. The present inventors have found that by performing the reaction described above, it is possible to suppress a decrease in the measured value due to competition between the antibody used for the immunoassay and the host-derived antibody, and to further increase the detection sensitivity, and thus completed the present invention.

That is, the present invention provides a human parvovirus B19 antigen, which comprises treating a specimen with a treatment solution containing at least one of the following surfactants (1) to (4) under acidic conditions of pH 3.5 or less. And a method for immunoassay.
(1) An amphoteric surfactant having an alkyl group having 10 or more carbon atoms and a quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and a 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 a steroid skeleton and quaternary ammonium in the molecule

  Further, 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 an immunoassay for an antigen protein of human parvovirus B19 having no envelope, even when a host-derived antibody such as a neutralizing antibody that competes with the antibody used for detection is present in the sample, the host The complex formed between the derived antibody and the antigen to be measured can be dissociated, and a decrease in the measured value can be prevented. 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 for which human parvovirus B19 antigen is to be measured is treated with a treatment solution containing a specific surfactant under acidic conditions of pH 3.5 or less. The surfactant used is at least one of the following (1) to (4).
(1) An amphoteric surfactant having an alkyl group having 10 or more carbon atoms and a quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and a 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 a 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] An amphoteric surfactant having the structure represented can be mentioned, and specific examples include the surfactants shown in Table 1 below, but are not limited thereto.

In the cationic surfactant (2), the alkyl group is preferably a straight-chain alkyl group. More preferably, the alkyl group has 12 or more carbon atoms. Preferred examples of the surfactant _{(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 done.

  In the nonionic surfactant (3), the alkyl group is preferably a straight-chain alkyl group. Preferred examples of the surfactant (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 specific examples (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 (1) and (2) described above.

Preferred examples of the amphoteric surfactant (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 this respect, the second embodiment has features common to the above-described preferred embodiment (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 (1) to (4), surfactants that can be preferably used include C12APS, C14APS, C16APS, C12TAB, C14TAB, C12TAC, and C14TAC, and in particular, C12APS in the present invention. 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 concentration, but it is preferable that the total concentration is about 10% or less.

  In the present invention, in addition to the above treatments with the surfactants (1) to (4), a polyoxyethylene alkylphenyl ether-based nonionic surfactant, and a polysorbate-based nonionic surfactant are further used. The sample processing by at least one selected may be performed. By using any of the surfactants (1) to (4) above in combination with these nonionic surfactants, the sensitivity of the immunoassay can be further increased. The concentration of these nonionic surfactants which can be used in combination in the treatment liquid may be the same as the surfactants (1) to (4). If these non-ionic surfactants are further added to the processing solution, the working process is simple.However, before mixing these non-ionic surfactants and the sample, the treatment with the above-mentioned processing solution is performed. These nonionic surfactants may be added after the treatment or after the treatment with the treatment solution.

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

  Polysorbate surfactants include polysorbate 20 (polyoxyethylene sorbitan monolaurate, trade name Tween20), polysorbate 40 (polyoxyethylene sorbitan monopalmitate, trade name Tween40), and 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) Tween 85) and the like. The polysorbate-based surfactant is also called a Tween-based surfactant or a polyoxyethylene sorbitan-based surfactant.

  Preferred specific examples of the polyoxyethylene alkylphenyl ether-based surfactant and the polysorbate-based surfactant include, but are not limited to, Triton X-705, polysorbate 20, and polysorbate 80.

  Alternatively, in addition to the above-described treatments with surfactants (1) to (4), sample treatment with a denaturant may be further performed. Specific examples of the denaturing agent include guanidine, guanidine salt, guanidine derivative, and urea. The sensitivity of the immunoassay can be further increased even when the surfactant and the denaturant of any of the above (1) to (4) are used in combination. Specific examples of the guanidine salt include, but are not limited to, guanidine hydrochloride, guanidine carbonate, guanidine nitrate, guanidine phosphate, guanidine sulfamate, and the like. Specific examples of the guanidine derivative include, but are not limited to, guanidinobenzoic acid, guanidinoglutaric acid, guanidinosuccinic acid, guanidinoacetic acid, guanidinopropionic acid, and guanidinobenzimidazole. The concentration of the denaturant in the treatment solution may be about 0.5M to 5M. A modifier may be further added to the treatment liquid, or the treatment with the modifier 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 treated (that is, a solution obtained by mixing the specimen and the treatment liquid) has a pH of 3.5 or less. The pH of the sample solution during the treatment may be, for example, about pH 3.0 or less, or about pH 2.8 or less. Acids that can be used to bring the sample solution under treatment to such acidic conditions are not particularly limited, and inorganic acids 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 bring the sample solution under treatment into an acidic condition, for example, an acidic buffer such as a glycine hydrochloride buffer or a citrate buffer adjusted to an appropriate pH may be used. An appropriate amount of an acid or acid buffer may be added to the treatment solution, or an acid or acid buffer may be mixed with the specimen separately from the treatment solution.

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

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

  After treating the specimen by mixing the treatment liquid and the specimen, the pH of the specimen solution is adjusted to near neutrality by neutralizing the specimen solution with an alkaline solution. Neutralization may be performed before the reaction with the antibody, or neutralization may be performed simultaneously with the reaction with the antibody. For example, by raising 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 sample with the antibody solution. it can.

The anti-human parvovirus B19 antibody used for the immunoassay for the human parvovirus B19 antigen in the treated sample may be a polyclonal antibody or a monoclonal antibody. Generally, monoclonal antibodies are preferably used. Further, instead of the antibody, an antigen-binding fragment such as Fab ′, F (ab ′) ₂ , scFv or the like can be used. Anti-human parvovirus B19 antibodies are known and are also used in commercially available kits. Such known antibodies can be preferably used. Alternatively, since polyclonal antibodies, monoclonal antibodies, and methods for producing antigen-binding fragments are well known, an anti-human parvovirus B19 antibody or an antigen-binding fragment thereof is prepared using human parvovirus B19 antigen as an immunogen, and immunoassay is performed. May be used. The human parvovirus B19 antigen protein used as an immunogen can be prepared, for example, with reference to JP-A-7-147986. Alternatively, it can also be obtained by extracting and purifying 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 in virus particles.

  The immunoassay for the human parvovirus B19 antigen in the present invention can be carried out by a known immunoassay such as a competition method or a sandwich method. Among them, the sandwich method can be preferably employed in the present invention. Specific examples of the sandwich immunoassay include chemiluminescent enzyme immunoassay (CLEIA), enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay; ELISA), radioimmunoassay, and electrochemiluminescence immunoassay. There are various methods. In the present invention, any method may be used.

  In a sandwich measurement system, one of two types of antibodies or antigen-binding fragments thereof is usually 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 treated sample treated according to the present invention, since the parvovirus B19 antigen is considered to be present mostly as a monomer, the solid-phase antibody and the labeled antibody used in the sandwich method recognize different sites of the 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 material of the solid phase 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 that can easily immobilize the antibody on its surface and can easily separate the immune complex formed during the measurement from unreacted components. In particular, a plastic plate or a magnetic particle used for an ordinary immunoassay is preferable. From the viewpoint of ease of handling, storage and separation, it is most preferable to use magnetic particles of the above-mentioned materials. The binding of the antibody to these solid phases can be performed by a conventional method well known to those skilled in the art.

  The labeling substance is not particularly limited, and the same labeling substance as used in known immunoassay systems can be used. Specific examples include enzymes, fluorescent substances, chemiluminescent substances, staining substances, radioactive substances and the like. Known enzymes such as alkaline phosphatase (ALP), peroxidase, and β-galactosidase can be used as the enzyme, but are not limited thereto.

  Briefly, the procedure of sandwich immunoassay is as follows. First, a treated sample is reacted with a solid phase antibody, washed, B / F separated, and then reacted with a labeled antibody. After washing again and B / F separation, signals (fluorescence, luminescence, color development, etc.) from the labeled antibody are detected. When an enzyme is used as a labeling substance, an appropriate substrate may be added and reacted, and a signal generated by the enzyme reaction may be detected. Immunoassay was performed on standard samples with known concentrations containing human parvovirus B19 antigen at various concentrations, and a calibration curve was prepared by plotting the correlation between the amount of signal from the label and the antigen concentration in the standard sample. By doing 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 using the two-step method, a one-step method in which the treated sample, the solid-phase antibody, and the labeled antibody are simultaneously reacted may be used.

  The sample subjected to the method of the present invention is typically a sample separated from a human. If the sample donor is infected with human parvovirus B19, the sample is not particularly limited as long as it is a sample expected to contain the antigen of the virus, such as serum, plasma, blood-derived samples such as blood for transfusion, Plasma fraction preparations and the like can be mentioned.

  The immunoassay kit for carrying out the immunoassay of the present invention comprises a sample treatment solution containing at least one of the above-mentioned surfactants (1) to (4), an anti-human parvovirus B19 antibody or its antigen-binding property. And fragments. The sample processing solution of the kit may contain an acid or an acidic buffer so that the pH of the sample solution being processed becomes the acidic condition as described above, or an acid solution such as an acidic buffer may be used for the sample processing. It may be contained separately from the liquid. In addition, the kit includes a denaturant as described above, and at least one selected from a polyoxyethylene alkylphenyl ether-based nonionic surfactant and a polysorbate-based nonionic surfactant in the sample processing solution. Further, it may be contained, or may be contained separately from the sample processing solution, and may be appropriately added and used at the time of 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 particles immobilized with anti-parvovirus antibody>
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 magnetized 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, the anti-parvovirus antibody-immobilized particles particles diluent (400 mM Tris buffer, 1 mM EDTA2Na, 0.1% NaN 3, 2.0% BSA, pH 9.0) was suspended in.

<Preparation of alkaline phosphatase-labeled antibody>
Desalted alkaline phosphatase (ALP) and N- (4-maleimidobutyryloxy) -succinimide (GMBS) (final concentration: 0.3 mg / mL) were mixed and left at 30 ° C. for 1 hour to perform maleimidation. Next, in a coupling reaction solution (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 1: 1 molar ratio. The mixture was mixed at a ratio and reacted at 25 ° C. for 1 hour. Superdex200 10/300 using column chromatography (GE Corp.), purification buffer (100 mM Tris buffer, 150 mM NaCl, 0.1% NaN 3, pH8.0) , the main flow rate 0.5 mL / min The peak was separated and purified to obtain an alkaline phosphatase-labeled antibody. At the time of measurement, the alkaline phosphatase-labeled antibody was suspended in a labeling solution (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 tank, and the mixture was stirred and incubated at 37 ° C. for 6.5 minutes. Thereafter, 50 μL of the antibody-immobilized particles were dispensed and stirred. Thereafter, the mixture was incubated at 37 ° C. for 8 minutes to perform B / F separation and washing. 50 μL of the enzyme-labeled antibody was dispensed into the reaction tank, and after stirring, the mixture was incubated at 37 ° C. for 8 minutes to perform B / F separation and washing. Then, a lumipulse substrate solution containing a chemiluminescent substrate, 3- (2'-spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) 200 μL was dispensed into the reaction tank, and after stirring, the mixture was incubated at 37 ° C. for 4 minutes, and the luminescence was measured with a luminometer. The actual measurement was performed with a fully automatic chemiluminescence 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 samples were 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. Samples were used Tris buffer Human purified inactivated antigens (50 mM Tris, 150 mM NaCl , 1 mM EDTA2Na, 0.1% NaN 3, 2.0% BSA, pH 7.2) to which was dissolved in a positive sample. In addition, a model obtained by adding an inhibitory antibody to a positive sample was used as a model of a sample containing an antibody used as a reagent and an antibody to be batting. 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 ratio (%) = (count value of positive sample to which inhibitory antibody was added) / (count value of positive sample to which inhibitory antibody was not added) × 100

  Table 5 shows the results. When the sample was treated with an acidic pretreatment solution to which C12TAB, C12APS, C14APS, and C16APS were added, the binding separation rate of the antibody was significantly improved.

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

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

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

  Table 7 shows the results. With a surfactant having 10 or more carbon atoms in the alkyl chain, the effect of improving the bond separation rate was observed. It was also found that a cationic surfactant also exhibited an effect.

4. Confirmation of effect of surfactant in 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. The sample diluting the sample to 10 ^{n (n} = 3~8) times in Lumipulse for dilutions were measured. The inhibitory antibody was used by mixing Antibodies 1 to 3, and was added so that the final concentration of the mixed antibody became 100 μg / mL. This antibody concentration is an amount that is not significantly different from the amount of specific IgG generated in human blood. The surfactant used was C12APS, and was added so as to be 2% in the pretreatment liquid. The pH of the sample solution during the pretreatment was adjusted to 2.4. In addition, a plurality of negative samples and positive samples were measured in advance under the condition that both the surfactant and the inhibitory antibody were not added, and the cutoff value was calculated from the obtained count value. In this measurement system, a positive sample can be determined if the count value is 5500 or more.

  Table 8 shows the results. The DNA titers in the table are the parvovirus DNA titers (IU / mL). When an inhibitory antibody is added, the sensitivity is reduced by about 100 times when no surfactant is added.On the other hand, when C12APS is added, the sensitivity is the same as that when no inhibitory antibody is added, even in the presence of the inhibitory antibody. Had. That is, in the presence of the inhibitory antibody, the sensitivity was increased 100-fold by the addition of the 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 CHAPS, an amphoteric surfactant, and MEGA8 and MEGA10, a nonionic surfactant, 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 one as described above was used.

  Table 9 shows the results. As for the nonionic surfactant, MEGA10 showed a remarkable improvement in the bond separation rate. With CHAPS (amphoteric surfactant), a remarkable effect was observed when antibody 3 whose binding site to parvovirus B19 butted with a labeled antibody was added.

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

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

7. Examination of effect of C12APS + denaturant
The effect of using a modifier combined with C12APS was evaluated. Urea (2M, 4M) and guanidine (4M) were used as denaturants. The pH during the pretreatment was 2.8. The sample is 6. The same one as described above was used.

  Table 11 shows the results. The use of C12APS in combination with a denaturing agent further improved the binding separation rate.

8. Investigation of surfactant concentration The concentration of the surfactant added to the pretreatment solution was examined. C12APS was used as a 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 one as described above was used.

  Table 12 shows the results. The bond separation rate was improved under all conditions from 0.1% to 2.0%. It is considered that an improvement effect can be obtained if the surfactant is present in the pretreatment liquid at a concentration of 0.1% or more. It was confirmed that the bond separation rate increased in a surfactant concentration-dependent manner.

9. (Comparative Example) Investigation 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 solution adjusted to have a pH of 2.4 during the pretreatment. The sample is 2. The same one as described above was used.

  Table 13 shows the results. When urea was used at a concentration of 4M or more, the count value was significantly reduced even in a positive sample. Addition of 2M urea, whose count value was maintained, did not show any effect of improving the bond separation rate.

Claims (12)

An immunoassay method for the human parvovirus B19 antigen, comprising treating a specimen with a treatment solution containing at least one of the following surfactants (1) to (4) under acidic conditions of pH 3.5 or less.
(1) An amphoteric surfactant having an alkyl group having 10 or more carbon atoms and a quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and a 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 a 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 (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 method according to any one of claims 1 to 5, further comprising treating the sample with at least one nonionic surfactant selected from a polyoxyethylene alkylphenyl ether surfactant and a polysorbate surfactant. The described method.   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 any one of claims 1 to 7, further comprising treating the sample 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. An immunoassay kit for human parvovirus B19 antigen, 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) An amphoteric surfactant having an alkyl group having 10 or more carbon atoms and a quaternary ammonium in the molecule
(2) Cationic surfactant having an alkyl group having 10 or more carbon atoms and a 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 a steroid skeleton and quaternary ammonium in the molecule At least one nonionic surfactant selected from a polyoxyethylene alkylphenyl ether-based surfactant and a polysorbate-based surfactant, or further contained in the sample processing solution, or separately from the sample processing solution. The kit of claim 11, comprising a kit.