JP5630866B2 - Method for detecting HIV - Google Patents

Description

  The present invention relates to a method for detecting human immunodeficiency virus (hereinafter referred to as HIV) by immunological measurement. More specifically, the present invention relates to a method for inactivating an antibody present in a sample, a method for detecting an HIV antigen containing the same, and a reagent used therefor as a specimen pretreatment for the HIV antigen detection method.

  HIV is a causative virus of Acquired Immune Deficiency Syndrome (hereinafter referred to as AIDS), which is classified in the retroviridae lentivirus genus. AIDS is an important public health disease recognized for the first time in 1981. To date, more than 60 million people have been infected with HIV and more than 20 million people have died from AIDS (non-patented) Reference 1). On the other hand, the number of infected people in Japan is just over 10,000, but the number continues to increase at an accelerated rate, and it is important to prevent the spread of infection and early diagnosis of latently infected people (Non-patent Document 2). ).

  In HIV diagnosis, HIV antibody screening test methods such as enzyme antibody method (ELISA), particle aggregation method (PA), immunochromatography method (IC), antibody confirmation test such as Western Blot method, fluorescent antibody method (IFA) and the like Tests such as HIV antigen test, antigen-antibody simultaneous test, virus isolation, and nucleic acid diagnostic method (PCR) are performed. In the HIV antibody test, a period (window period) until infection can be confirmed after infection is 3 to 4 weeks. Therefore, an HIV antigen test that can shorten this period is useful (Non-patent Document 3).

  Nucleic acid diagnostics (PCR), one of the HIV pathogenetic tests, plays a major role in the above-mentioned diagnosis, testing of blood for blood transfusion, evaluation of AIDS treatment effect by antiviral agents, and decision of treatment policy. Yes. However, since the nucleic acid diagnostic method is expensive and complicated to operate, a method capable of detecting HIV antigen with high sensitivity by a cheaper and simpler antigen test is desired.

  On the other hand, in the HIV antigen test, the HIV antigen is detected at the early stage of infection, but when the immune system is subsequently activated and antibodies are produced, the antigen is generally not detected. This is because the produced antibody and the antigen form an immune complex, which makes it impossible to measure the antigen, and it is considered that the antigen can be measured by dissociating the immune complex and inactivating the antibody.

  As a method for inactivating HIV antibody, a method of boiling in Triton (registered trademark) X-100 solution (Non-patent Document 4), a method of inactivating with a glycine-HCl buffer (Non-patent Document 5), and the like have been performed. It was. However, boiling in Triton X-100 solution inactivates the antibody while denaturing the antigen, and inactivation with glycine-HCl buffer is insufficient to inactivate the antibody. This method also has a problem that the measured value is low.

   On the other hand, reducing agents such as mercaptoethanol and dithiothreitol have been known to have an action of reducing disulfide bonds of proteins and maintaining thiol groups in a reduced form, and are used as protein protective agents. However, no example of using a reducing agent in the pretreatment for HIV antigen measurement has been reported for the purpose of suppressing antigen destruction by heat treatment.

AIDS Epidemic Update.December 2009. (http://data.unaids.org/pub/Report/2009/JC1700_Epi_Update_2009_en.pdf) Katsuji Teruya, Clinical and Virus 36 (4), 247-256, 2008 Takako Shima et al., Journal of Infectious Diseases 81 (5), 562-572, 2007 Ruengpung Sutthent, et al., Journal of Clinical Microbiology, Mar. 1016-1022, 2003 Denis R. Henrard, et al., Journal of Clinical Microbiology, Jan. 72-75, 1995

  The present invention relates to a method for inactivating an HIV antibody before immunologically measuring an HIV antigen made in view of the above-described situation, and effectively suppresses the HIV antibody while maintaining the stability of the antigen. It aims at providing the method to inactivate and the reagent used therefor.

  As a result of intensive studies to solve the above problems, the present inventor, in a pretreatment liquid in which a compound having a thiol group as a reducing agent or a phosphine is added to an acidic amino acid buffer containing a nonionic surfactant. It was found that by heat-treating the sample, the antibody can be effectively inactivated while maintaining the stability of the HIV antigen. Furthermore, the present inventor has found that it is effective to reduce the concentration of each reducing agent by combining reducing agents in order to solve the problem that the sample precipitates and solidifies when the reducing agent concentration is increased. It was. The present invention has been made based on these findings.

That is, the present invention has the following configuration.
(1) In immunological measurement, the sample is heat-treated in a pretreatment solution in which a compound having a thiol group and / or phosphine is added to an acidic amino acid buffer containing a nonionic surfactant. A pretreatment method for HIV antigen detection.
(2) The pretreatment method for HIV antigen detection according to (1), wherein the heat treatment temperature of the sample is 56 ° C. or higher.
(3) The pretreatment method for HIV antigen detection according to (1) or (2), wherein the compound having a thiol group is 2-diethylaminoethanethiol or a salt thereof, or 2-mercaptoethanol.
(4) The pretreatment method for HIV antigen detection according to any one of (1) to (3), wherein the phosphine is tris (2-carboxyethyl) phosphine or a salt thereof.
(5) The pretreatment method for HIV antigen detection according to any one of (1) to (4), wherein the pH of the acidic amino acid buffer is 1.0 or more and 2.5 or less.
(6) The pretreatment method for HIV antigen detection according to any one of (1) to (5), wherein the nonionic surfactant is an ether type or an ester type.
(7) A method for immunological measurement of HIV antigen, comprising the following steps.
(A) a step of performing the pretreatment method according to any one of (1) to (6);
(B) A step of detecting the presence or absence of HIV antigen using a probe that binds to HIV antigen, or a step of quantifying HIV antigen using a probe that binds to HIV antigen.
(8) The immunological measurement method according to (7), wherein the probe is an anti-HIV antibody.
(9) A pretreatment solution for a sample containing HIV, comprising the following (a) to (c):
(A) nonionic surfactant (b) acidic amino acid buffer (c) a compound having a thiol group and / or a phosphine (10) a compound having a thiol group is 2-diethylaminoethanethiol or a salt thereof, or 2- The pretreatment liquid according to (9), which is mercaptoethanol.
(11) The pretreatment liquid according to (9) or (10), wherein the phosphine is tris (2-carboxyethyl) phosphine or a salt thereof.
(12) The pretreatment liquid according to any one of (9) to (11), wherein the nonionic surfactant is an ether type or an ester type.
(13) A reagent for detecting the presence or absence of HIV antigen, comprising the pretreatment liquid according to any one of (9) to (12), and further comprising a probe that binds to HIV antigen Reagent kit.
(14) The reagent or reagent kit according to (13), wherein the probe is an anti-HIV antibody.
(15) A reagent or reagent kit for quantifying HIV antigen, comprising the pretreatment liquid according to any one of (9) to (12), and further comprising a probe that binds to HIV antigen .
(16) The reagent or reagent kit according to (15), wherein the probe is an anti-HIV antibody.

  With the HIV detection method of the present invention or the reagent used therefor, it is possible to inactivate HIV antibodies while maintaining the stability of the antigen, and it is possible to measure HIV antigens with high sensitivity and accuracy.

  The present invention relates to a pretreatment for detection of HIV antigen by immunological measurement in a solution in which a compound having a thiol group as a reducing agent and / or phosphine is added to an acidic amino acid buffer containing a nonionic surfactant. A heat treatment of the sample is performed.

  The antigen to be detected in the present invention is not particularly limited as long as it is all or part of the protein encoded by the HIV gag gene. Examples of HIV-1 antigen include p24 and p17, and examples of HIV-2 antigen include p26 and p16.

  The reducing agent used in the present invention is used for the purpose of maintaining the stability of the antigen by suppressing the destruction of the HIV antigen by treatment with a high temperature and a surfactant. Any reducing agent may be used, and 2-mercaptoethanol (hereinafter referred to as 2-ME), a compound having a thiol group, 2-diethylaminoethanethiol or a salt thereof (hereinafter referred to as DEAET), or Tris (2-carboxyethyl) phosphine or a salt thereof (hereinafter referred to as TCEP) is suitable. In particular, by combining 2-ME and TCEP, the concentration of each reducing agent can be suppressed, and the stability of HIV antigen can be maintained to the maximum while suppressing precipitation and coagulation of the sample. The concentration of the reducing agent used is preferably 0.001M or more and 0.12M or less as the concentration in the sample pretreatment liquid. More specifically, it is preferable that 2-ME is 0.01M to 0.1M, DEAET is 0.04M to 0.12M, and TCEP is 0.001M to 0.002M.

  In the sample pretreatment of the present invention, inactivation of the antibody is insufficient at low temperatures, but when the treatment time is increased at high temperatures, the sample in the reaction solution is likely to precipitate or coagulate during measurement. Therefore, the treatment temperature and time need to be within a range that can sufficiently inactivate the antibody while suppressing precipitation and coagulation of the sample in the reaction solution. Although the effect of the present invention can be obtained by setting the temperature to 56 ° C. or more and 100 ° C. or less, suitable treatment time varies depending on the temperature. A person skilled in the art can set the processing temperature and time with reference to the embodiment of the present invention. As shown in the Examples, when the temperature is 56 ° C. or higher and 65 ° C. or lower, the treatment time is preferably 1 hour or longer, and more preferably 1 hour or longer and 3 hours or shorter. In the vicinity of 70 ° C., the treatment time is preferably 30 minutes or more, and more preferably 30 minutes or more and 1 hour or less. Further, in the vicinity of 97 ° C., it is preferably 5 minutes or more and 15 minutes or less, and more preferably 10 minutes or more and 15 minutes or less.

  The nonionic surfactant used in the present invention is used to effectively inactivate HIV antibodies by combining with heat treatment. Any ether type nonionic surfactant or ester type nonionic surfactant can be used without particular limitation. Examples of ether types include polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene alkylphenyl ethers, and ester types include sorbitan fatty acid esters, sucrose fatty acid esters, glycerin fatty acid esters, poly Examples include glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyethylene glycerin fatty acid ester, and polyethylene glycol fatty acid ester. The HLB value is preferably 12 or more and 18 or less, and more preferably 13 or more and 18 or less. The concentration of the surfactant used in the present invention is preferably 0.5% or more and 10% or less as the concentration in the sample pretreatment liquid.

  The amino acid used in the present invention is used as a buffer for the sample pretreatment solution for the purpose of further enhancing the effect of maintaining the stability of HIV antigen. It is not particularly limited as long as it is an amino acid, glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine Or modified amino acids such as hydroxyproline and hydroxylysine. The concentration used is preferably 0.05 M or more and 0.3 M or less, more preferably 0.1 M or more and 0.2 M or less, and further preferably 0.1 M or more and 0.15 M or less as the concentration in the sample pretreatment solution.

  The sample pretreatment liquid of the present invention is adjusted to an acidic pH at which the sample in the reaction solution hardly precipitates and coagulates during measurement and can effectively dissociate the HIV antigen from the antibody. The pH of the sample pretreatment solution is preferably 1.0 or more and 2.5 or less, more preferably 1.5 or more and 2.0 or less, and further preferably 1.75 or more and 2.0 or less.

  The HIV antigen measurement method after the specimen pretreatment of the present invention is not particularly limited as long as it is an immunoassay method. Various methods such as enzyme immunoassay (EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), colloidal gold agglutination, immunochromatography, latex agglutination (LA), immunoturbidimetric (TIA) Applicable to the method.

  Probes such as antibodies can be used for HIV antigen measurement after the specimen pretreatment of the present invention. The animal species or clone from which the antibody is derived is not particularly limited as long as it can detect the HIV antigen. Examples include antibodies derived from rabbits, goats, mice, rats, guinea pigs, horses, sheep, camels, chickens, etc. Monoclonal antibodies or polyclonal antibodies may be used. Also, all subclass antibodies suitable for specific binding to HIV antigens can be used. Of course, it is also possible to use fragments such as recombinant antibodies, Fab, Fab ′ or F (ab ′) 2 fragments.

  The sample used for the specimen pretreatment of the present invention is not particularly limited as long as it is a sample that may contain an HIV antigen. Examples include serum, plasma, whole blood, urine, saliva, lymph, cell tissue extract, and other biological specimens.

  Examples of the present invention are shown below, but the present invention is not limited to these examples.

(Examination of reducing agent 1)
Using a specimen obtained by adding HIV antigen to HIV-negative human serum, the type and concentration of the reducing agent contained in the specimen pretreatment solution were changed, and the degree of suppression of antigen inactivation by specimen pretreatment was examined.

(1) Solid-phase antibody A commercially available anti-HIV-1 p24 antibody (Meridian c65690M, c65941M) or anti-HIV-2 p26 antibody (AUSTRAL Biologicals H12A-851-5, Fitzgerald 10-001401) was diluted with PBS. , Magnetic particles activated according to the method described in the instruction manual of the product (Dynabeads (registered trademark) M-280-T Tosylactivated manufactured by invitrogen) were added and stirred at 25 ° C. for 1 hour, and then 0.5% BSA (oriental yeast) The mixture was stirred at 25 ° C. for 1 hour to obtain a solid-phase antibody solution.

(2) Luciferase-labeled antibody Streptavidin introduced with a maleimide group was reacted with the anti-HIV-1 p24 antibody or anti-HIV-2 p26 antibody obtained by reduction to obtain a thiol group according to a standard method. This was reacted with biotinylated luciferase (manufactured by Kikkoman), fractionated and purified using Superdex (registered trademark) 200 (manufactured by GE Healthcare Bioscience), diluted 100-fold with PBS containing 0.5% casein Na and used.

(3) Luminescent reagent 1, 2
HCl was added to Tris (hydroxymethyl) aminomethane (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a 50 mM Tris-HCl pH8.5 aqueous solution. Luminescent reagent 2 was prepared by adding D-luciferin to 0.47 mM, ATP · Mg to 2 mM, and sulfuric acid Mg to pH 6.4, 20 mM ADA (Donjin Chemical) buffer solution.

(4) Sample pretreatment solution
Various reducing agents shown in Table 1 below were added to 0.875% Triton X-100 (polyoxyethylene isooctyl phenyl ether), 0.1 M glycine-HCl buffer pH 2.0.

(5) Sample pretreatment
A sample prepared by adding 10 ng / ml of p24 antigen (Meridian R18301) or p26 antigen (AUSTRAL Biological H12A-850-5) to HIV negative serum (SCANTIBODY LABORATRY) was used as a sample. 400 μl of the specimen pretreatment solution (4) was added to 100 μl of this sample, and after stirring, heated in a 97 ° C. water bath for 10 minutes and then cooled to obtain a pretreated specimen.

(6) Immunological measurement method 200 μl of the pretreated specimen of (5) was weighed in an immune reaction tube, and 200 μl of a neutralizing solution (0.7 M Tris-HCl pH 8.5) was added. At this time, as a control, 40 μl of a sample that had not been pretreated was separately weighed into an immunoreaction tube, and operations other than the addition of a neutralizing solution were performed in the same manner. 20 μl of the immobilized antibody solution prepared in (1) was added to each immunoreaction tube and reacted at 37 ° C. for 15 minutes (first reaction). After the reaction, 1 ml of a washing solution (PBS + 0.05% Tween (registered trademark) 20 (polyoxyethylene sorbitan monolaurate)) was added, and the immobilized antibody was magnetized with a magnet to remove the reaction solution. Further, 1 ml of the washing solution was added and stirred, and then the solid-phased antibody was similarly collected with a magnet to remove the washing solution. After repeating this operation three times, 50 μl of luciferase-labeled antibody was added instead of the washing solution, and the mixture was reacted at 37 ° C. for 15 minutes (second reaction). After the reaction, 1 ml of the washing solution was added, and the immobilized antibody was collected with a magnet to remove the reaction solution. After adding 1 ml of the washing solution to this and stirring, similarly, the immobilized antibody was collected by a magnet to remove the washing solution. After repeating this operation three times, 100 μl of luminescent reagent 1 was added instead of the washing solution, and 100 μl of luminescent reagent 2 was further added. It was measured.

(7) Results Tables 2 to 5 show the emission intensity and the ratio (%) of the emission intensity at each reducing agent concentration with respect to the emission intensity without pretreatment.

  From these results, the addition of the reducing agent to the pretreatment solution suppresses the inactivation of the HIV antigen due to the pretreatment (Tables 2 to 4), and the higher the concentration of each reducing agent, the higher the HIV antigen. Although the deactivation suppression effect tends to increase (Tables 2 and 3), it was shown that the higher the concentration of the reducing agent, the easier it is to clot due to insolubilization of proteins and the like in serum (Tables 2 to 4). On the other hand, when a pretreatment solution containing 2-ME and TCEP as a reducing agent is used, particularly in the p26 antigen, a high HIV antigen deactivation inhibitory effect that cannot be obtained with a pretreatment solution containing a single reducing agent, that is, It was shown that there is an effect of maintaining the stability of the antigen (Table 5). Compared to p24 antigen, preactivation of p26 antigen has a greater degree of antigen inactivation, which is due to the fact that p24 antigen is a native antigen while p26 antigen is a recombinant antigen. It is thought that there is. Although described in the margins of each table, those marked with “*” at the end of the numbers in the table are poorly collected due to the solidification of the immobilized antibody due to coagulation of the reaction solution, and the measurement is performed well. Indicates no.

(Examination of reducing agent 2)
Using the HIV-1 positive specimen as a sample, the effect of the pretreatment method of the present invention was examined.
In all of the following examples up to Example 9, the operations for preparing the immobilized antibody, the luciferase-labeled antibody, and the luminescent reagents 1 and 2 were performed in the same manner as in Example 1.

(1) Sample pretreatment solution
Various reducing agents shown in Table 6 below were added to 0.875% Triton X-100, 0.1 M glycine-HCl buffer pH 2.0. As a control, a pretreatment solution without a reducing agent was also prepared.

(2) Sample pretreatment Using commercially available HIV-1 antibody-positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution of (1) to 100 μl of this sample, stir, and then heat in a 97 ° C water bath for 10 minutes And then cooled.

(3) Immunological measurement method The immunological measurement method was performed in the same manner as in Example 1 (6), except that the measurement of the untreated sample (control) was not performed.

(4) Results Table 7 shows the luminescence intensity and the ratio (%) of the luminescence intensity at each reducing agent concentration to the luminescence intensity when 0.001M TCEP and 0.1M 2-ME are added as reducing agents.

  When any reducing agent is used, the luminescence intensity is remarkably increased compared to the case where no reducing agent is added, and the HIV antibody can be effectively inactivated while suppressing the destruction of the HIV antigen and maintaining the antigen stability. It was shown that. In particular, it was shown that the highest emission intensity was obtained when 2-ME and TCEP were contained as reducing agents.

(Comparison with conventional method)
Using HIV-1 positive specimen as a sample, the method of the present invention (Method A), the conventional method (Method B (PerkinElmer Life Sciences kit), Method C (ZeptMetrix kit), Method D (Non-patent Document 4), Method E (Nonpatent literature 5)) The measured value after the pre-processing by was compared.

(1) Sample pretreatment liquid and sample pretreatment Method of the present invention Sample pretreatment solution (0.875% Triton X-100, 0.1M glycine-HCl buffer pH 2.0, 0.1M 2-ME, 0.001M TCEP) in 100 μl of commercially available HIV-1 antibody positive serum (ProMedDx) (Hydrochloric acid salt-containing) 400 μl was added and stirred, and then treated in a 97 ° C. water bath for 10 minutes. After cooling, 200 μl was weighed and neutralized by adding 0.7 M Tris-HCl pH 8.5.
B. PerkinElmer Life Sciences kit method Add 108 μl of sample pretreatment solution (24 μl of 5% Triton X-100, glycine reagent (containing 1.5 M glycine) 108 μl) to 108 μl of commercially available HIV-1 antibody positive serum (ProMedDx) Then, it processed at 37 degreeC for 60 minutes. Thereafter, 108 μl of Tris reagent (containing 1.5 M Tris) was added for neutralization.
C. ZeptMetrix Kit Method 105 μl of a sample pretreatment solution (containing glycine-HCl buffer) was added to 105 μl of commercially available HIV-1 antibody-positive serum (ProMedDx), followed by treatment at 37 ° C. for 60 minutes. Thereafter, 105 μl of a base reagent (containing Tris-HCl and sodium azide) and 35 μl of Lysing Buffer were added for neutralization.
D. Method of Non-Patent Document 4 To 100 μl of commercially available HIV-1 antibody positive serum (ProMedDx), 600 μl of sample pretreatment solution (0.5% Triton X-100) was added and stirred, and then treated at 100 ° C. for 5 minutes. After cooling, it was used as a sample for immunoassay.
E. Method of Non-Patent Document 5 190 μl of sample pretreatment solution (0.15 M glycine-HCl buffer pH 2.0) is added to 100 μl of commercially available HIV-1 antibody positive serum (ProMedDx) and stirred for 10 minutes at 70 ° C. Processed. After cooling, 10 μl of 3.5 M Tris-HCl was added for neutralization.

(2) Immunological measurement method 400 μl of the sample prepared by the method A in (1), 129 μl of the sample prepared by the method B in (1), 137 μl of the sample prepared by the method C in (1), 280 μl of the sample prepared by the method D in (1) and 120 μl of the sample prepared by the method E in (1) were sampled and immunologically measured. (All sampling amounts were adjusted to be equivalent to 40 μl of HIV-1 positive serum.) A sample obtained by diluting a commercially available HIV-1 antibody positive serum (ProMedDx) 10 times with PBS without pretreatment and A sample prepared by adding 10 ng / ml of p24 antigen (Meridian R18301) to HIV negative serum (SCANTIBODY LABORATRY) and diluting 10 times with PBS was used as a control. Except for the sampling amount of each sample, the emission intensity was measured in the same manner as in Example 1 (6).

(3) Results Table 8 shows the measurement values (luminescence intensity) by each pretreatment method and the PCR method HIV-RNA measurement values (described in the product information attached when purchasing HIV-1 positive serum).

  In each of the A to E methods, the measured value was higher than the result without pretreatment, but the measured value of the specimen No. 4 was the method of the present invention (the A method) in the B method, the C method, and the E method. ) And about 1/3 of the method of the present invention (Method A). From this, specimen No. 4 is resistant to antibody breakage, so method B (Triton X-100, treated at 37 ° C in glycine reagent), method C (treated at 37 ° C in glycine-HCl buffer), method E ( The antibody was not sufficiently inactivated in the glycine-HCl buffer solution at 70 ° C, and the antibody D was inactivated and the antigen was denatured in the method D (treated in Triton X-100 at 100 ° C). It is inferred that the measured value is lower than that of the method of the invention. A similar tendency was observed for Sample No. 1. On the other hand, specimens No. 2 and No. 3 had no significant difference in measured values except for the D method, and the D method had a lower value than the other methods, so that these specimens easily inactivate antibodies. For this reason, antibody inactivation was sufficiently achieved by the B, C, and E methods. However, in the D method, the antibody was inactivated and the antigen was denatured. As a result of this study, the method of the present invention (Method A), which obtained the highest measured values in all of samples No. 1 to No. 6, was effective in protecting the antigen while sufficiently inactivating the antibody. It was shown that the detection sensitivity can be significantly higher than that of the conventional method because it can be maintained.

(Examination of treatment temperature and time 1)
HIV-1 positive specimens were used as samples, and the treatment temperature and time were changed to examine the effects of antibody inactivation and antigen stability retention.

(1) Sample pretreatment solution A sample pretreatment solution having the following formulation was used.
・ 0.875% Triton X-100
・ 0.1M glycine-HCl buffer solution pH2.0
・ 0.1M 2-ME
・ 0.001M TCEP (hydrochloride)

(2) Sample pretreatment Using commercially available HIV-1 positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution (1) to 100 μl of this sample, and after stirring, 37 ° C, 50 ° C, 70 ° C or After heating at 97 ° C. for 10 minutes, 30 minutes, 1 hour, 3 hours or 21 hours, it was cooled.

(3) Immunological assay The same method as in Example 1 (6) was used.

(4) Results Table 9 shows the luminescence intensity and the ratio (%) of the luminescence intensity of each pretreatment condition to the luminescence intensity at 97 ° C. for 10 minutes.

  Assuming that the luminescence intensity at 97 ° C for 10 minutes was 100%, the luminescence intensity in the range of 100 ± 15% was obtained for all specimens at 70 ° C for 30 minutes and 1 hour. At other temperatures and treatment times, the emission intensity was out of the range of 100 ± 15% in some or all samples. Of the specimens used for the study, No. 11, 12, and 13 showed a tendency to inactivate antibodies less than No. 14 and 15, but specimen No. 11 was the least susceptible to inactivation of antibodies. At 70 ° C. for 10 minutes, the emission intensity was 61.8% (when the emission intensity at 97 ° C. for 10 minutes was taken as 100%).

(Examination of treatment temperature and time 2)
HIV-1 positive specimens were used as samples, and the treatment temperature and time were changed to examine the effects of antibody inactivation and antigen stability retention.

(1) Sample pretreatment solution A sample pretreatment solution having the following formulation was used.
・ 0.875% Triton X-100
・ 0.1M glycine-HCl buffer solution pH2.0
・ 0.1M 2-ME
・ 0.001M TCEP (hydrochloride)

(2) Sample pretreatment Using commercially available HIV-1 antibody-positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution (1) to 100 μl of this sample, and after stirring, 56 ° C, 60 ° C or 65 ° C For 30 minutes, 1 hour, 3 hours or 21 hours and then cooled. As a control, a heat treatment at 97 ° C. for 10 minutes was performed.

(3) Immunological assay The same method as in Example 1 (6) was used.

(4) Results Table 10 shows the luminescence intensity and the ratio (%) of the luminescence intensity of each pretreatment condition to the luminescence intensity at 97 ° C. for 10 minutes.

  Assuming that the luminescence intensity at 97 ° C for 10 minutes is 100%, the luminescence intensity within the range of 100 ± 15% is obtained for all specimens at 56 ° C, 60 ° C and 65 ° C for 1 hour and 3 hours. However, the luminescence intensity was out of the range of 100 ± 15% in some samples or all samples at other treatment times. Of the specimens used in the study, specimen No. 31 is the least resistant to inactivation of the antibody. In 30 minutes at 56 ° C, 60 ° C, or 65 ° C, the luminescence intensity at 97 ° C for 10 minutes is 100%. The emission intensity was less than 85%.

(Surfactant study 1)
Using HIV-1 positive samples as samples, the effect of inactivating antibodies and maintaining the stability of antigens was examined by changing the type of surfactant added to the sample pretreatment solution.

(1) Sample pretreatment solution
Various surfactants shown in Table 11 below were added to 0.1M glycine-HCl buffer solution pH 2.0, 0.1M 2-ME, 0.001M TCEP (hydrochloride). As a control, a pretreatment liquid without addition of a surfactant was also prepared.

(2) Sample pretreatment Using commercially available HIV-1 positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution (1) to 100 μl of this sample, and after stirring, heat in a 97 ° C water bath for 10 minutes , Cooled.

(3) Immunological measurement method The immunological measurement method was performed in the same manner as in Example 1 (6), except that the measurement of the untreated sample (control) was not performed.

(4) Results Table 12 shows the emission intensity and the ratio (%) of the emission intensity when each surfactant is added to the emission intensity when 0.875% Triton X-100 is added.

  SDS, an anionic surfactant, and C12TAB, a cationic surfactant, when the emission intensity when using a pretreatment solution with Triton X-100, a nonionic surfactant added, is 100% Alternatively, most of the luminescence intensity was 85% or less when using a pretreatment liquid to which C12APS as a zwitterionic surfactant was added. From this, it was suggested that a nonionic surfactant is suitable as the surfactant to be added to the pretreatment liquid.

(Surfactant study 2)
Using HIV-1 positive samples as samples, the effect of inactivating antibodies and maintaining the stability of antigens were examined by changing the type and concentration of nonionic surfactant added to the sample pretreatment solution.

(1) Sample pretreatment solution
Various surfactants shown in Table 13 below were added to 0.1M glycine-HCl buffer solution pH 2.0, 0.1M 2-ME, 0.001M TCEP (hydrochloride). As a control, a pretreatment liquid without addition of a surfactant was also prepared.

(2) Sample pretreatment Using commercially available HIV-1 positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution (1) to 100 μl of this sample, and after stirring, heat in a 97 ° C water bath for 10 minutes , Cooled.

(3) Immunological assay The same method as in Example 1 (6) was used.

(4) Results Tables 14 and 15 show the emission intensity and the ratio (%) of the emission intensity when each surfactant was added to the emission intensity when 1.0% Triton X-100 was added.

  In Table 14, when the luminescence intensity when using the pretreatment liquid to which Triton X-100 is added is 100%, ether type Emalex 620 (polyoxyethylene stearyl ether), Triton X-305 (polyoxyethylene) The luminescence intensity when using the pretreatment solution to which isotonyl phenyl ether), Triton X-405 (polyoxyethylene isooctyl phenyl ether) and ester type Tween 20 were added was 85 except for specimens Nos. 41 and 43. The emission intensity when using a pretreatment solution to which ester ether type Emalex GM-40 (polyoxyethylene glyceryl monostearate) was added was a sample at an Emalex GM-40 concentration of 0.5%. All except for No. 44 and 45 were 85% or less. Specimens No. 41 and 43 are considered to be specimens in which antibodies are not easily inactivated. From the results shown in Table 14, it was shown that an ether type or ester type nonionic surfactant is suitable as the surfactant to be added to the pretreatment liquid. Tables 14 and 15 show that the effect of the present invention can be obtained by setting the addition concentration to 0.5% to 10%.

(Examination of buffer solution)
Using HIV-1 positive samples as samples, the effect of inactivating antibodies and maintaining the stability of antigens was examined by changing the type of buffer contained in the sample pretreatment solution.

(1) Sample pretreatment solution
0.875% Triton X-100, 0.1M 2-ME and 0.001M TCEP (hydrochloride) with 0.1M glycine, glutamine, arginine, citric acid, KCl, serine, valine, methionine, histidine, proline, tryptophan or glutamic acid Then, the pH was adjusted to 2.0 with HCl to obtain a sample pretreatment solution.

(2) Sample pretreatment Using commercially available HIV-1 antibody positive serum (ProMedDx) as a sample, add 400 μl of the sample pretreatment solution (1) to 100 μl of this sample, stir, and then heat in a 97 ° C water bath for 10 minutes And then cooled.

(3) Immunological measurement method The immunological measurement method was performed in the same manner as in Example 1 (6), except that the measurement of the untreated sample (control) was not performed.

(4) Results Tables 16, 17-1 and 17-2 show the luminescence intensity and the ratio (%) of the luminescence intensity when each buffer solution is contained to the luminescence intensity when the glycine-HCl buffer solution is contained.

  When citric acid-HCl and KCl-HCl buffers were used, the measurement was poor due to cloudiness and coagulation of the reaction solution, but in the case of buffers containing amino acids, both were equivalent to glycine-HCl buffer. It was shown that there is an effect of maintaining the antibody inactivation and antigen stability.

(Recovery rate of added antigen)
The p24 antigen was added to HIV-1 positive specimens, and the measured antigens were compared before and after the antigen addition and with or without pretreatment to examine the recovery rate of the added antigen.

(1) Sample pretreatment solution A sample pretreatment solution having the following formulation was used.
・ 0.875% Triton X-100
・ 0.1M glycine-HCl buffer solution pH2.0
・ 0.1M 2-ME
・ 0.001M TCEP (hydrochloride)

(2) Sample pretreatment Using commercially available HIV-1 antibody positive serum (ProMedDx) and HIV negative serum (SCANTIBODY LABORATRY) as a sample, add 400 μl of sample pretreatment solution (1) to 100 μl of this sample. After stirring, the mixture was heated in a 97 ° C. water bath for 10 minutes and then cooled. Further, 10 μl of p24 antigen (Meridian R18301) 200 ng / ml was added to 90 μl each of the above HIV-1 positive serum and HIV negative serum, and pretreatment was similarly performed at 97 ° C. for 10 minutes.

(3) Immunological assay The same method as in Example 1 (6) was used.

(4) Results Luminescence intensity, ratio of luminescence intensity of each sample to the luminescence intensity when p24 antigen was added to HIV negative serum (control) (%) and PCR method HIV-RNA measured value (when purchasing HIV-1 positive serum) Table 18 shows the information described in the attached product information.

  When an antigen was added to an HIV-1 positive sample and measured as it was, it was not detected or very low except for one case (Sample No. 48). On the other hand, when specimen pretreatment is performed, the recovery rate of antigen is 75.2-84.5% when HIV negative specimen without pretreatment is used as a control, and 89.6-100.6% when HIV negative specimen with pretreatment is used as a control. there were. From this, it was shown that the antibody was almost inactivated by the pretreatment, while the antigen stability was maintained.

  The specimen pretreatment method of the present invention and the reagent used therefor can effectively inactivate the antibody while maintaining the stability of the antigen in HIV antigen detection by immunological measurement, so that the antibody is produced after infection. It is possible to measure HIV antigen with high sensitivity and high accuracy. Therefore, in addition to the diagnosis of the presence or absence of HIV infection, it can be applied to evaluation of antiviral drug treatment effects that require a highly sensitive antigen measurement method, qualitative tests, and quantitative tests for blood for transfusion.

Claims (16)

  In the immunological measurement, the sample is heat-treated in a pretreatment solution in which a compound having a thiol group and / or phosphine is added to an acidic amino acid buffer containing a nonionic surfactant, Pretreatment method for HIV antigen detection.   The pretreatment method for HIV antigen detection according to claim 1, wherein the heat treatment temperature of the sample is 56 ° C or higher.   The pretreatment method for HIV antigen detection according to claim 1 or 2, wherein the compound having a thiol group is 2-diethylaminoethanethiol or a salt thereof, or 2-mercaptoethanol.   The pretreatment method for HIV antigen detection according to any one of claims 1 to 3, wherein the phosphine is tris (2-carboxyethyl) phosphine or a salt thereof.   The pretreatment method for HIV antigen detection according to any one of claims 1 to 4, wherein the pH of the acidic amino acid buffer is 1.0 or more and 2.5 or less.   The pretreatment method for HIV antigen detection according to any one of claims 1 to 5, wherein the nonionic surfactant is an ether type or an ester type. A method for immunological measurement of HIV antigen, comprising the following steps.
(A) performing the pretreatment method according to any one of claims 1 to 6;
(B) A step of detecting the presence or absence of HIV antigen using a probe that binds to HIV antigen, or a step of quantifying HIV antigen using a probe that binds to HIV antigen.   The immunoassay method according to claim 7, wherein the probe is an anti-HIV antibody. A pretreatment solution for a sample containing HIV, comprising the following (a) to (c):
(A) Nonionic surfactant (b) Acidic amino acid buffer (c) Compound having a thiol group and / or phosphine   The pretreatment liquid according to claim 9, wherein the compound having a thiol group is 2-diethylaminoethanethiol or a salt thereof, or 2-mercaptoethanol.   The pretreatment liquid according to claim 9 or 10, wherein the phosphine is tris (2-carboxyethyl) phosphine or a salt thereof.   The pretreatment liquid according to claim 9, wherein the nonionic surfactant is an ether type or an ester type.   A reagent or a reagent kit for detecting the presence or absence of an HIV antigen, comprising the pretreatment liquid according to any one of claims 9 to 12, and further comprising a probe that binds to the HIV antigen.   The reagent or reagent kit according to claim 13, wherein the probe is an anti-HIV antibody.   A reagent or reagent kit for quantifying HIV antigen, comprising the pretreatment liquid according to any one of claims 9 to 12, and further comprising a probe that binds to HIV antigen.   The reagent or reagent kit according to claim 15, wherein the probe is an anti-HIV antibody.