WO1995034812A1

WO1995034812A1 - METHOD OF ASSAYING SPECIFIC IgM ANTIBODY AND REAGENT THEREFOR

Info

Publication number: WO1995034812A1
Application number: PCT/JP1995/001191
Authority: WO
Inventors: Toru Yoshimura
Original assignee: Dainabot Co., Ltd.
Priority date: 1994-06-16
Filing date: 1995-06-15
Publication date: 1995-12-21
Also published as: JP3487643B2; JPH085633A

Abstract

The invention provides a method of detecting a specific IgM in an infectious disease at an acute stage, e.g., a HAV-related antibody, a method of permitting a wider-range assay by reducing the predilution factor, a method of improving the detection percentage in various diseases such as hepatitis A, and a method of diagnosing and detecting the diseases without fail. By diluting a specimen with at least IgM or an aqueous solution thereof, it becomes possible to conduct favorably an immunoassay of a specific IgM antibody against a target antigen present in the specimen by using an antihuman IgM antibody reagent. More particularly, a specific IgM antibody can be assayed by (1) diluting, if necessary, a specimen with a buffer, a diluent or an aqueous diluent solution and then with at least IgM or an aqueous solution thereof, then reacting a specific IgM antibody contained in a specimen with an antihuman IgM antibody-binding solid carrier to effect an immunological reaction between the IgM antibody in the specimen and the solid-phase antihuman IgM antibody, and (2) conducting either (a) an immunological reaction of the obtained reaction product with a specified antigen reagent and then with a labeled antibody against the antigen reagent, or (b) an immunological reaction of the obtained reaction product with a specified labeled antigen reagent.

Description

Description Specific IgM antibody assay and reagents

The present invention provides a method for immunologically measuring a specific IgM antibody against a target antigen in a test sample using an anti-human IgM antibody reagent, wherein the test sample comprises at least IgM antibody. Alternatively, the present invention relates to a specific IgM antibody measuring reagent characterized by being diluted with an aqueous solution containing IgM, and a measuring method using the reagent. Background art

Immunoassays are widely used in human clinical tests and disease diagnosis, as well as in animal clinical tests and disease diagnosis, and in the analysis, measurement and quantification of a wide range of other analytes. It is applied in fields such as detection and detection. This immunoassay utilizes an antigen-antibody reaction between an antigen and an antibody against the antigen, but usually, the detection of the occurrence of this antigen-antibody reaction can be visually determined, Or, since it is difficult to detect, various methods have been developed to make the detection and detection easy. Of these, the measurement of specific IgM antibodies generated by in vivo immune reactions, especially in the acute phase, has been attracting attention because it is useful for diagnosis of early infections such as viral infections and pathogen infections. I have. However, the specific IgM antibody has a problem that its measurement is difficult because it is interfered by rheumatoid factor or the like or interfered by IgG or the like. A typical example of the immunoassay using the IgM measurement is an IgM antibody capture assay, such as hepatitis A virus. (Hepatitis A vi ru s: HAV) Infection diagnosis, Hepatitis B virus core antigen (HBc), rubella, measles, mumbus, etc. . In particular, hepatitis A virus (HAV) was discovered in the feces of patients with acute hepatitis A by Feinstone and others in 1973, and in 1977 the proliferation of experimentally infected chimpanzee liver tissue was observed. After the report, the growth of primary marmoset hepatocytes and fetal monkey fetal kidney cells was reported in 1979, and HAV-cultured cell lines have been used for primary and established africana monkey kidney cells and human diploid cells. It has been reported that it grows in such as.

By the way, in Japan, although the incidence of hepatitis A virus infection is decreasing, the number of anti-HAV antibody-negative people is increasing, mainly in young people, while the age of the elderly is positive. Preventing HAV infection because adults are more likely to come into contact with foods and drinks contaminated with HAV due to their high distribution, and adults are more likely to develop hepatitis once infected. In that sense, there is a need for accurate and rapid detection of HAV infection. In addition, since HAV is transmitted mainly by oral transmission via feces and the like, the risk of infection in areas with poor environmental hygiene is high, and the chances of overseas travel have recently increased, thus testing HAV infection. It is also considered important to prevent transmission between relatives and employees. To detect HAV infection in the acute phase, anti-HAV antibodies that appear in the patient's blood due to a strong immune response in vivo following HAV infection, particularly IgM antibodies specific to HAV, are detected. The following IgM antibody capture assay using the HAV antigen immunologically reactive with this anti-HAV (IgM) antibody as a reagent has been developed. Typical Ig

Diagnosis of acute hepatitis A infection according to the M antibody capture assay Using an anti-human IgM antibody with specificity for one of the above chains, a solid phase coated with the anti-human IgM antibody (// single-chain specific antibody), for example, a solid phase carrier as shown below, was measured. The reaction is performed by reacting with a sample, then reacting with an HAV antigen reagent, and finally reacting with a labeled anti-HAV antibody. However, in such an acute phase, the concentration of specific IgM such as anti-HAV (IgM) antibody and total IgM to be measured in test samples such as blood, serum, and plasma vary. In general, it is known that the total amount of IgM in blood is usually about 0.4 to 2 mgZm1, but the solid phase coated with the anti-human IgM antibody in the first reaction described above Since the amount of antibody may be insufficient, there is a problem that it is necessary to perform a pre-dilution of the test sample, for example, a high-magnification pre-dilution. Conventionally, the test sample was pre-diluted with a buffered aqueous solution, physiological saline solution, or the like. This will reduce the amount of total IgM antibody ^ The ratio of specific IgM to total IgM antibody cannot be reduced. Therefore, there is a problem that it is difficult to obtain a necessary measurement range. In addition, in an automated measurement system, there was a problem that the amount of diluent was limited, and a problem that the measurement range was limited.

As mentioned above, specific IgM, such as HAV-related antibodies, can be detected earlier, or the pre-dilution dilution can be reduced to allow for a wider range of measurements. Reliable measurement methods in the area are urgently and strongly required. Disclosure of the invention

The present inventors can easily set the required measurement range of the test sample without the above-mentioned problems, detect specific IgM, for example, HAV-related antibody at an earlier stage, or perform pre-dilution. Reduce the dilution ratio to obtain a wider range of measurements. As a result of diligent research to find a measurement method suitable for an automated measurement system that enables measurement, it was found that these problems could be solved by a simple method, and the present invention was completed.

The present invention provides a method for immunologically measuring a specific IgM antibody against a target antigen in a test sample using an anti-human IgM antibody reagent, the method comprising: Alternatively, the present invention provides a specific IgM antibody measurement reagent characterized by being diluted with an aqueous solution containing IgM, and a measurement method using the reagent. In a more specific embodiment, the invention provides that the sample is diluted once with an aqueous solution of a buffer, diluent or diluent, and then the sample is diluted with at least IgM or an aqueous solution containing IgM. After dilution, an IgM antibody having specificity for a specific antigen, such as an anti-virus-specific IgM antibody, in the sample is reacted with a solid support coated with anti-human IgM antibody, etc. The IgM antibody in the sample is immunologically reacted with the solid-phase anti-human IgM antibody, and (a) a specific antigen reagent such as virus is immunologically reacted. (B) reacting a labeled antigen, such as a virus, with a specific antigen reagent with a labeling agent, or reacting it immunologically with a labeled antibody such as a chemiluminescent-labeled antiviral antibody. Method for measuring specific IgM antibody and reagents used in the method It is provided. By adding human IgM, it is possible to achieve a wider range of measurement, and to simplify the preparation of the measurement sample, such as setting the measurement range such as adjusting the sample concentration. Facilitates application in immunoassay systems. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the relationship between sample concentration and luminescence. Detailed description of the invention The present invention relates to a method for immunologically measuring a specific IgM antibody against a target antigen in a test sample using an anti-human IgM antibody reagent, wherein the test sample contains at least IgM or IgM. Provide a specific IgM antibody measurement reagent characterized by being diluted with an aqueous solution, and a measurement method using the reagent. Examples of the measurement target sample used in the present invention include biological materials such as whole blood, serum, plasma, saliva, and biological mucus. In particular, whole blood, serum, and plasma are preferably used as a sample to be measured.

In the present invention, an IgM antibody specific to a target antigen is added to a sample by adding an aqueous solution of a human IgM-containing fraction or purified human IgM to the sample without being affected by the amount of IgM in the sample. To be measured. About 0.

In the case of an aqueous solution containing 005% of human IgM, the aqueous solution will increase the sample by a factor of about 10-500, preferably about 50-200, more preferably about 70-150, most preferably about 80-150. It may be diluted up to 120 times. By adding human IgM, it is possible to achieve a wider range of measurement, and it is possible to easily set the measurement range, such as preparation of the measurement sample, for example, adjustment of the sample concentration, and automated immunoassay. It is easier to apply in systems.

The sample to be measured can be used by adding a buffer, a diluent, a chelating agent, a preservative, and the like before the above treatment, if necessary. The sample may optionally be diluted 50- to 500-fold. Examples of the buffer, diluent or diluent include water, phosphate or phosphate buffer, tris (hydroxymethyl) aminomethane (Tris) buffer, sodium chloride such as physiological saline, N — (2-Hydroxitytyl) piperazine-N,-(2-ethanesulfonic acid) (HEPES) solution, piperazine-N, N, monobis (2-ethanesulfonic acid) (PI BES) solution, 3 — (Cyanohexylamino) 1-propanesulfonic acid (CAPS) solution, 3- (morpholino) propa Sulfonic acid (MOPS) solution, N, N'-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES) solution, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) solution N- (2-acetoamide) -1-aminoethanesulfonic acid (ACES) solution, amino acid solution and the like. These can be used alone or in any combination. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis (yS-aminoethylether) -N, N, N ', N'-tetraacetic acid (EGTA). These chelating agents can be used in the range of about 0.01 mM to about 20 mM.

The sample can be diluted with an aqueous solution containing hepatitis-negative human serum, plasma, and human IgM, if necessary. In a more specific embodiment, the present invention provides a method wherein the sample is once diluted with buffer, diluent or an aqueous solution of a diluent to some extent, and then diluted with at least IgM or an aqueous solution containing IgM. An IgM antibody having specificity for a specific antigen, such as an anti-virus-specific IgM antibody in the sample, is reacted with a solid phase carrier coated with an anti-human IgM antibody to react the IgM antibody in the sample. (A) Next, a specific antigen reagent such as a virus is immunologically reacted, and the obtained reaction product is reacted with a chemiluminescent-labeled anti-virus antibody or the like. A specific Ig comprising reacting a labeled antibody immunologically, or (b) immunologically reacting a labeled antigen obtained by labeling a specific antigen reagent such as a virus with a labeling agent. M antibody measurement method and reagents used for the measurement method are provided. It is.

An example of a particularly preferable measurement system is a method for diagnosing acute hepatitis A infection by measuring the blood of a patient with an anti-HAV antibody of the IgM type, which occurs in the acute phase of HAV infection. This IgM type anti-HAV antibody is In a system for differential measurement, // a solid phase coated with a single-chain specific anti-human IgM antibody, for example, a solid support as shown below, is reacted with the sample to be measured, and then a HAV antigen reagent is used. The reaction is carried out, and finally the reaction is carried out sequentially with the labeled anti-HAV antibody.

As the HAV antigen reagent used in the system for specifically measuring an IgM type anti-HAV antibody according to the present invention, a virus antigen obtained by an in vitro cell culture method is used. It includes an HAV extract isolated from a cell lysate obtained by lysing infected cells or a cell derived therefrom. The HAV extract was obtained from, for example, African green monkey kidney culture cells, human liver tumor cell line PLCZPRF / 5, Hep.

A HAV-infected cell that can be cultured, and more preferably a cell line cell capable of producing HAV in large quantities, is transformed into a known growth medium, such as Eagle's minimum essential medium (Eagle's MEM). Minimum essential medium (Dulbecco's MEM), PRM 1-1640 (Gibco). Eag1e's MEM), N- (2-hydroxyxethyl) piperazine-N, 1- (2- Ethanesulfonic acid) (HE PES) Buffered Eagle MEM, Phosphate buffered L-15-a medium, Hanks 'solution (Hanks' banced saltsolution), etc., as needed. Culture in fetal serum (FCS), antibiotics such as pencilin, trebutomycin, yeast extract, pactopeptone, lactalbumin hydrolyzate, and other cell growth factors, and then It is obtained as follows from the obtained cell culture.

That is, the nutrient medium is removed from the cell culture obtained as described above, and the cells are then buffered with physiological saline, phosphate, or the like, and if necessary, a chelating agent such as EDTA is used. Wash with the addition of The harvested cells are typically isolated by a chelating agent such as EDTA. And polyoxyethylene ether (typically available under a trade name such as 0.5% Triton X-100) and buffered with phosphate containing non-ionic surfactants. Lysate in a buffered solution, such as a phosphate buffered solution containing ImM EDTA and 0.5% Triton X-100, or a phosphate buffer containing dexcholate The cell lysate thus obtained is incubated, if necessary, for example, for about 10 to 15 minutes, and then centrifuged, for example, for about 1,000 to 20, OOO xg, preferably Centrifugation at about 2,000 to 10,000 Xg for about 5 to 60 minutes, preferably about 10 to 30 minutes can obtain an HAV extract. Thus, hepatitis A virus is obtained by removing the nucleus-derived substances, cell organelles, and crushed substances from the cell lysate by centrifugation. HAV extracts can also be obtained, for example, as described in US Pat. No. 4,721,675. The HAV extract can be further purified, if necessary, by, for example, a black form extraction method, an enzyme treatment method, or a sucrose gradient centrifugation method.

The HAV extract thus obtained is then subjected to a treatment for inactivating its infectivity by a known method or a modified method thereof. The inactivation treatment is performed, for example, by treatment with a formalin solution, for example, incubation at about 37 ° C under a dilution of about 1: 3000 to 1: 7000 in a about 25-45% formalin solution, for example, under a dilution of 1: 4000. However, other appropriate methods can be selected from known methods and applied. This process may be performed, for example, for two weeks, and may be shorter or longer. In the treatment liquid for this treatment, a buffer, a diluent or a diluent, a chelating agent, a preservative, and the like can be added as necessary. Buffers, diluents or diluents include water, phosphate buffer, tris (hydroxymethyl) aminoaminomethane (Tris) buffer solution, sodium chloride solution such as physiological saline, N- (2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) (H EPES) solution, piperazine-N , N'-bis (2-ethanesulfonic acid) (PI BES) solution, 3- (cyanohexylamino) 1-propanesulfonic acid (CAPS) solution, 3- (morpholino) propanesulfonic acid (MO

PS) solution, amino acid solution and the like. These may be used alone or in any combination. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis-β-aminoethyl ether) Ν, Ν, Ν ′, N′-tetraacetic acid (EGTΑ).

According to the present invention, the thus obtained HAV extract in which the infectivity has been inactivated can be used as it is as a HAV antigen, or can be further treated with a surfactant. These surfactant-treated HAV extracts can be used as preferred.c As the surfactant, an appropriate surfactant can be selected from known or commercially available ones. Surfactants are suitable.

Examples of the anionic surfactant include an alkali metal salt or an earth metal salt of a higher fatty acid having 12 to 18 carbon atoms such as potassium stearate; an alkali metal salt of bile acid; Organic fatty acid salts of higher fatty acids, such as triethanolamine, fatty acids of 18 to 18 carbon atoms, such as lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS), sulfuric acid esters of higher fatty acids or higher alcohols, alkyl sulfonates, and alkyls Examples thereof include alkyl aryl sulfonates such as sodium benzene sulfonate, and LDS and SDS are particularly effective. Alkali metals include sodium, potassium, lithium and the like, and alkaline earth metals include potassium, magnesium and the like. These surfactants coexist It is preferable to select the amount to be used according to the amount of protein to be used.

It can be used in the range of 0.01% vΖv to about 10% v / v. Particularly preferably, using SDS, about 0.05% v / v to about 5% v / v, and more preferably about 0.5% v / v to about 1% in the absence of other coexisting proteins. It can be used in the range of 0% v / v. When treating the HAV extract with a surfactant, dilute the HAV extract with a buffer, diluent or diluent as necessary, and mix or suspend with a surfactant solution that provides the required concentration. . The mixture thus obtained can be agitated if necessary. In some cases, glass beads or the like may be added to the mixture, followed by stirring. As long as the stirring process can improve the measurement sensitivity, for example, only gentle mixing can be performed, or vigorous stirring mixing can be performed. The treatment can be performed at room temperature, under cooling, or at a temperature of 37 ° C. or higher as long as the measurement sensitivity can be improved. The HAV extract treated with the surfactant can be used as it is for the next processing, or it can be used once for storage and then used for the next processing. It can be used for the next process after washing and other treatments according to the requirements. These treatments can be selected so as to suppress non-specific adsorption during measurement and improve sensitivity.

In the treatment solution for the surfactant treatment of the present invention, a buffer, a diluent, a diluent, a chelating agent, a preservative, and the like can be added and used. Buffers, diluents or diluents include water, phosphate or phosphate buffer, Tris buffer, for example, sodium chloride solution such as saline, HEPES solution, PI BES solution, CAPS solution, MOPS solution, N, N 'Monobis (2-hydroxyxethyl) -2-aminoethanesulfonic acid (BES) solution, N-Tris (hydroxymethyl) methyl-1-aminoethanesulfonic acid (T ES) solution, N- (2-acetoamide) _2-aminoethanesulfonic acid (ACES) solution, amino acid solution and the like. These can be used alone or in any combination. Examples of the chelating agent include EDTA, EGTA and the like. According to the present invention, the HAV extract is optionally treated with the above surfactant before inactivating its infectivity,

'Next, the obtained HAV treated with the surfactant may be inactivated by a known method or a modified method thereof. The inactivation treatment may be performed in the same manner as described above.

It can be performed by incubation treatment at a dilution of 000. In a more specific embodiment, the HAV antigen reagent used in the present invention comprises an HAV extract obtained from a cell lysate obtained by an in vitro cell culture method of about 0.5% / ¥ to about 1%. Mix with sodium dodecyl sulfate (SDS) solution at a concentration in the range of 0% v / v and then, if necessary, incubate at room temperature for 3 hours, for example, and treat 303 extract It can also be provided by gaining. In the present invention, a reagent for immunoassay of HAV antibody in a sample using the obtained SDS-treated HAV extract and an antibody in a sample using the same in combination with at least a step of treating an IgM or an aqueous solution containing IgM. Are also provided.

In measuring the specific IgM antibody in the present invention, methods such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, and chemiluminescence immunoassay can be used. In particular, chemiluminescence immunoassays, for example

The 1 gM capture solid phase chemiluminescence immunoassay is the preferred method because it allows for automated measurements. In the present invention, in measuring a specific IgM antibody in a sample, antigens and antibodies participating in an antigen-antibody reaction may be used, if necessary, for example, agar, agarose, cellulose, paper, nitrocellulose, dextran. , Gelatin, chitin, collagen, cotton, etc. Are polymers derived from natural products, acrylyl resins such as polystyrene, polyethylene, polyvinyl alcohol, and polyacrylamide, ion exchange resins, optical bridge resins, synthetic polymers such as Teflon and polyacetal, glass beads, silica gel, alumina, ceramic, Fine particles, beads, microplates, microtiter wells, microtubes, trips, membranes, trays, gels, etc., made of inorganic materials such as carbon and magnesium sulfate, as well as solid carriers such as red blood cells, latex particles, and emulsions The immobilized carrier is brought into contact with a sample containing an antibody or the like to be analyzed, and the antigen immobilized on the immobilized carrier and the antibody or the like in the analysis sample are specifically bound and reacted. Analyte specifically bound Can be detected.

In a preferred embodiment, in the present invention, the anti-human IgM antibody-bound solid carrier capable of reacting with a sample, such as polystyrene beads or polystyrene microparticles, can be used as the particulate carrier. In addition, any antibody can be used without particular limitation as long as it is an antibody against human IgM. Antibodies can be obtained by conventional methods.For example, Shigeru Muramatsu, et al., Experimental Biology Lecture 14, Immunobiology, Maruzen Co., Ltd., Showa 60, Japan Biochemical Society Ed. Immunochemical Chemistry Research Method, Tokyo Chemistry Dojin, 1996, ed., Biochemical Society of Japan, New Chemistry Experiment Course 12, Molecular Immunology III, Antigen 'Antibody' Capture, Tokyo Chemical Dojin, 1992, etc. For example, they can be obtained by immunizing a horse, a horse, a horse, a sheep, a goat, a goat, a rat, a mouse, or the like, or can be a monoclonal antibody. Alternatively, these can be arbitrarily used in combination. These antibodies may be digested with enzymes such as pepsin and papain, if necessary, and used as F (ab ') ₂ and Fab. As the anti-human IgM antibody, preferably, an antibody that specifically reacts with the / z chain, It is needless to say that an anti-chain-human IgM antibody may be used, and these may be a monoclonal antibody obtained by using cell fusion technology using mouse myeloma cells. In addition, the anti-HAV antibody may be one obtained from human HAV-positive serum or plasma having a high titer. Needless to say, a monoclonal antibody may be used as described above. Anti-H

B e antibody can be obtained from a virus culture, but can also be a recombinant antigen expressed in E. coli, yeast, etc. using genetic recombination <ε> ο

Radio I Takeno Atsu Si, enzyme immunoassay, fluorescence immunoassay, in chemiluminescent immunoassay, radioactive materials, such as ^{1 2 5} I, ³ ^Η, horseradish peroxide Okishida Ichize, β-ϋ one-galactosidase Antigens such as alkaline phosphatase, fluorescent dyes such as fluorescein, chemiluminescent dyes such as acridinium esters, and colored substances such as colloidal gold, colloidal selenium, or colored latex particles In some cases, a secondary antibody is used as a reagent, and specifically reacts with the antibody, complex, etc. in the analysis sample, and its radioactivity, enzyme activity, chemiluminescence, or the presence or absence of color is measured. It is possible to determine whether or not an antibody or the like was present in the sample. In the present invention, in particular, a chemiluminescent labeling method, for example, an ataridinium ester or a fluorescent labeling method, for example, a fluorescent or chemiluminescent immunoassay using a secondary antibody reagent labeled with luminescent lanthanide, etc., can perform automated measurement. This is the preferred method. In particular, a chemiluminescence immunoassay using a secondary antibody reagent labeled with an acridinium ester is preferable because it can perform automated measurement. Examples of the acridinium esters include, for example, JP-A-62-39598, JP-A-62-196969, and JP-A-63-575772. JP, JP-A-63-101-368, JP-A-633-1

1 2 564, JP-A-1-1999949, JP-A-11-261 No. 461, JP-A-2-966567, JP-A-2-133334, JP-A2-50332, JP2_50 No. 1 772, European Patent Application No. 0 82 636, UK Patent Specification 1, 461, 877, U.S. Patent Specification 3, 539, 5 N-alkyl or aryl acrylidinum 9 described in No. 74, etc. In particular, 10-alkyl-N-alkyl or arylsulfonyl-N described in JP-A-63-112564, U.S. Pat. No. 3,539,574, etc. —Alkyl or arylsulfonycrydinidium 9-carboxamide, N-methylacridinium-1-hydroxyrubonate and the like are mentioned as typical chemiluminescent labels. In the case of acridinium labeling, trigger one reagent treatment before measurement, for example, hydrogen peroxide, for example, an aqueous solution of about 0.01% to about 0.1% hydrogen peroxide, and sodium hydroxide, for example, about 0.1 N After treatment with an aqueous solution of about 0.5 N sodium hydroxide, the measurement can be performed using a luminometer or the like. Examples of the luminescent lanthanide include, for example, European Patent Application No. 0 6 8

875, U.S. Pat. No. 4,374,120, U.S. Pat. No. 4,283,382, U.S. Pat. No. 4,259,31 No. 3, U.S. Patent Specification No. 4,352,751, U.S. Patent Specification No. 4,432,907, European Patent Application No. 0 2013355, etc. Those which can chelate the luminescent lanthanide having the described aminocarbonic acid group can be mentioned. Also, the measurement can be facilitated by performing an excitation process using a laser beam or the like before the measurement. Of course, the labeling agent is not limited to the above, but may be appropriately selected from those known to be used in the relevant field, taking into account the equipment and location used for the measurement. Can be used. In order to bind or adsorb a solid carrier, a particulate carrier, a label, etc. to an antigen, an antibody, etc., a method generally used in the relevant field is used. For example, it can be performed by physical adsorption or chemical bonding such as ionic interaction, hydrophobic interaction, and covalent bond. For example, crosslinking agents include glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-0-phenylenediimide, N-succinimidyl 3- (2- Pyridyldithio) propionate, N-succinimidyl S-acetylmercaptoacetate, N-succinimidyl 4-1 (N-maleidomethyl) cyclohexane-11-carboxylate, N-succinimidyl 6-maleimide hexanoate, N-succinimidyl 4-acetylacetylaminobenzoate, N-succinimidyl 3- (p-hydroxyphenyl) propionate,

N-succinimidyl m-maleimidobenzoate, N-succinimidyl 4-maleimidobutyrate, N-succinimidyl (P-maleimidophenyl) acetate, N-succinimidyl 4 -— (ρ-maleimidphenyl) petitate And the like.

Examples of solid carriers and particulate carriers include those described above, for example, agar, agarose, cross-linked agarose, cross-linked alginic acid, cross-linked guar gum, cellulosic esters such as ditrocellulose and carboxycellulose, or mixed cellulose. Ester, gelatin, crosslinked gelatin, latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, Modification of natural or synthetic materials such as polyesters such as polyglycidyl methacrylate and acrolein-ethylene glycol dimethacrylate copolymer, polyamides, polyurethanes, and polyepoxy resins; Glass, such as activated glass, silica gel, kaolin, talc, silica, etc., such as unmodified polymerized carbohydrates, polymerized hydrocarbons, and their crosslinked derivatives —A material selected from the group consisting of inorganic materials such as alumina, alumina, barium sulfate and the like is used in the form of porous gels, fine particles, etc. In the present invention, the measurement is performed by competitive atssays, sandwich atsieties, It can also be carried out in a manner suitable for neutral attraction, solid-phase at- sity, chromatogram atssay, and the like. In the present invention, as a labeling reagent,

4-hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine, etc. and horseradish peroxidase, umbelliferyl galactoside, nitrophenylgalactoside, etc. and β-D-galactosidase, umbelliferyl Phosphate, nitrophenyl phosphate, NADΡ and other enzyme reagents such as alkaline phosphatase, glucose-6-phosphate and dehydrogenase, radioactive material reagents, fluorescein isothiocyanate, tetramethyl rhodamine isothiosine Fluorescent reagent, luminescent reagent, chemiluminescent reagent, colloid labeling reagent such as gold colloid, silver colloid, selenium colloid, magnetic reagent, etc. using acridinium ester, luminescent lanthanide, etc. Hap of biotin-labeled anti-biotin antibody Or the like can be used down-labeled anti-hapten antibody detection system reagent. As described above, in the present invention, the use of a chemiluminescent labeling reagent, for example, an acridinium ester or a fluorescent labeling reagent, for example, a secondary antibody reagent labeled with a luminescent lanthanide, can automate the measurement. preferable. In the measurement system of the present invention, a surfactant, a buffer, a diluent or a diluent, a blocking agent, a chelating agent, a preservative and the like other than those described above can be used. Preferred surfactants include polyoxyethylene sorbitan (typical is available under trade names such as Tween 20), polyoxyethylene ether (typical is Triton X-1 (Available under trade names such as 0 0), octylphenol-ethylenoxide condensate (typically N 0 nidet P-40, etc.).

Examples of the buffer, diluent or diluent include water, phosphate buffer, Tris buffer, physiological saline, and the like, such as HE PES solution, PBES solution, CAPS solution, MOPS solution, and amino acid solution. No. These can be used alone or in any combination. E as a chelating agent

DTA, EGTA and the like. Examples of the preservative include sodium azide, ethylparaben and the like. In addition, the measurement system of the present invention includes various animal sera, for example, sera serum, sera serum albumin (BSA), sera fetal serum (FCS), goat serum, egg white albumin, gelatin, and various milk proteins. For example, skim milk, casein, casein hydrolyzate, whey protein, and the like, and polyvinyl alcohol, polyvinylpyridone, and other substances selected from the group consisting of strength and the like can be added.

In the present invention, the reagent may be contained in a single container or a plurality of containers, and may be used after being mixed for use.

In a more specific embodiment of a typical measurement system for HAV infection diagnosis of IgM antibody measurement, the present invention provides a method of diluting a sample to an appropriate concentration with a physiological saline or the like, for example, about 80 to 120 times, It is preferably diluted about 100-fold, and then reacted with an appropriate amount of a human IgM solution and an anti-human IgM antibody-bound solid carrier or particulate carrier, and then (1) collected from HAV-infected cultured cells. The harvested HAV extract or (2) the HAV extract is immunologically reacted with at least a HAV antigen obtained by treating with a surfactant, for example, SDS, and the obtained reaction product is subjected to chemiluminescence. HAV characterized by immunologically reacting a labeled anti-HAV antibody, for example, an anti-HAV antibody labeled acridium, and reacting with a trigger reagent comprising a hydrogen peroxide solution and a sodium hydroxide solution, followed by detection. Methods for measuring antibodies may be provided.

Using viral antigens obtained by the in vitro cell culture method, When measuring an antibody that is immunologically reactive with the antigen, the HAV extract once isolated from a cell lysate obtained by lysing infected cells is further treated with a detergent. When used as an HAV antigen reagent, the measurement sensitivity can be significantly increased without unexpectedly adversely affecting antigen activity.

In the present invention, it can be used for a known immunological assay for measuring a specific IgM antibody, and is considered to be applicable to, for example, a specific antibody assay system caused by a virus infection, a pathogenic microorganism infection, or the like. Can be Viruses include herpes simplex, herpes zoster, varicella virus, mumbus, measles, rubella, AIDS virus (HIV), hepatitis B virus (HBV),

Hepatitis C virus (HCV) and the like, and pathogenic microorganisms include diphtheria bacteria (Corynebacteium diphtheriae), tetanus bacteria (Clostr idium tetani), cholera bacteria (Vibrio cholerae), botulinum bacteria (Clos iridium botul inum, Uenoles bacteria (Clostridium perfringens li, Staphylococcus aureus), for example, MRSA, Shigella (

Shigel la dysenteriae), B. pertussis (Salmonel la typhi), Paratyphi A (Salmonel la paratyphi), S. enteritidis (Salmonel la enteri tidis), Mycobacterium tuberculos, etc. Example

Next, the present invention will be described more specifically with reference to examples. However, it should be understood that the present invention is not limited to these specific examples and can be implemented in various forms as long as the idea is followed. It is.

Example 1 Preparation of IgM

Commercially available human IgM solution (Chemicon, USA) prior to use in a 0.021 \ Tris buffer (pH 8.5) containing 0.1% sodium azide prior to use. Dilute to give IgM reagent.

Preparation of anti-human IgM antibody coated microparticles

A polyclonal antibody (Jackson Immon Research Laboratories, USA), which has specificity for the human IgM chain obtained from goats (Jacks Immon Research Laboratories, USA.) ) Was bound to lipoxylated polystyrene latex fine particles (Ceradyne, USA; Seradyn, USA; 0.2 urn) by the method described below. First, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC; 16 mg / m 1) in 0.015 M MES (2- [N-morpholino] ethanesulfonate) buffer (PH4.7) was added. A polyclonal antibody anti-human IgM antibody (16 Omg / ml) was bound for 1.5 hours at room temperature. Next, washing was carried out using a 0.05 M phosphate buffer (pH 7.2) containing 1% Tween 20 and 0.9% NaCl. Ultimately, a 0.05 M Tris buffer containing 0.05% gelatin, 0.1% Tween 20, 0.9% NaCl and 0.1% sodium azide. (PH 7.4). The coated microparticles were diluted with a storage buffer such that the solid content% became 0.0625%, and used as an anti-human IgM antibody-coated microparticle reagent.

Preparation of acridinium-labeled anti-HAV antibody

3-alanine acridinium (lmg) was dissolved in anhydrous dimethylformamide (DMF) (100 z 1) and N-hydroxysuccinimide (NHS) (5.75 mg / m K 50 〃1) and EDAC (9.6 mg / mK501) were added successively, and the mixture was activated by stirring at 25 ° C for 48 hours in the dark. Protein A purified monoclonal anti-HA Activated vaccine in 0.1 M phosphate buffer (pH 8.0) containing 0.9% NaCl and 0.5% CHAPS containing V antibody (1 mgZml) Then, the reaction mixture was stirred at room temperature for 10 minutes. Buffer solution was 0.1 M phosphate buffer containing 0.1% CHAPS, 0.1% sodium azide and 0.9% NaCl (pH

6.3), the preparation was centrifuged, and the supernatant was equilibrated with the same buffer solution as the one after the replacement. Biosil SEC 250 (Bio-Rad, USA [Bio 1ad, USA. The fractions (1 ml) were analyzed by UV spectroscopy at 369 nm and 280 nm to determine the amount of acridinium bound. Store at about 4 ° C in concentrated fractions (about 100 / g / ml) and use 1% sodium caseinate, 0.1% tween 20, 0.1% sodium azide, 5 mM EDTA and 0.1% prior to use. The mixture was diluted with a 0.05 M phosphate buffer (pH 6.3) containing 9% NaCl to obtain an acridinium-labeled anti-HAV antibody reagent.

Preparation of HAV antigen

HAV was cultured using Bars Alexander cells (Barth—Alex and erCe11s) in a nutrient medium. Cultured cells were treated with a 0.02 M phosphate buffer (pH 7.2) containing 0.5% Triton X-100 and containing 5 mM EDTA and 0.9% NaCl.

), Stirred at 36 ° C for 16-68 hours, centrifuged, discarded the supernatant, and added the HAV extract to a formaldehyde dilution ratio of 1: 4000. Stirring at 3 ° C for 3 days inactivated the infectivity of HAV. The inactivated HAV extract was 1% bovine serum albumin, 0.05% Tween 20, 0.1% sodium azide, 5m

0.1 M phosphate buffer containing M EDTA and 0.9% NaC1 The resulting solution was diluted with a solution (pH 7.2) to obtain an HAV antigen reagent. HAV antigen reagents were stored at 4 till use.

Secret

The HAVAB-M positive channel sample was diluted with physiological saline. The diluted sample was further diluted 5-fold with IgM reagent (301).

For comparison, HAVAB-M negative panel sample was diluted with physiological saline, and then diluted 5-fold with the buffer used for diluting IgM. C Diluted sample (1 25 // 1) Was added to the container, and anti-human //-IgM antibody-coated fine particle reagent (301) was added thereto, and reacted at 37 for 20 minutes.c.The reacted fine particles were captured by a glass fiber filter. The plate was washed twice with a 1 M boric acid buffer solution (pH 8.5) (300/1). Next, an HAV antigen reagent (30〃1) was added to the surface of the filter, and allowed to react with the fine particles captured on the surface of the filter at 37 ° C for 20 minutes. The filters were washed once with 0.1 M borate buffer (pH 8.5) (1001) and once with the same buffer (300 ^ 1). Next, an ataridium-labeled anti-HAV antibody reagent (30 に 1) was added to one surface of the filter, and allowed to react with the fine particles trapped on the surface of the filter for 10 minutes at 37 ° C. The filter was washed once with 0.1 M borate buffer (pH 8.5) (1001) and once with the same buffer (300 z1).

Transfer this filter to a chemiluminescence reader where 0.25N N

The trigger solution (50 // 1) containing 0.4% hydrogen peroxide in & 011 was sent to the filter. Ataridium bonded to the fine particles emitted light, and the amount of generated light was measured. At low HAVAB—M positive, ° N sample concentrations, the dilution with the IgM reagent gives an emission of 2136 (photon count) and 13299 without the addition of the IgM reagent (photon count), Moderate HAVAB—at concentrations of M-positive panel samples, The dilution yields 5010 luminescence (photon count) and 16788 luminescence (photon count) without the addition of IgM reagent. High HAVAB-M positive panel sample concentration is 1253 for dilution with IgM reagent. The luminescence of 1 (photon count) was 18029 without the addition of IgM reagent (photon count). By the way, when the SDS-treated HAV antigen was used as in Example 2 below (IgM reagent added), the luminescence (photon count) was 4381 for the low panel, 1107 for the medium panel, and 1007 for the high panel. Met. Example 2 Preparation of 308 treatment

HAV was cultured using bar Alexander cells (Barth-AlexandeCells) in a nutrient medium. The cultured cells were mixed with a 0.02M phosphate buffer (pH 7.2) containing 0.5% Triton X-100 and containing 5 mM ED and 0.9% NaCl, After stirring at 36 ° C for 16-68 hours, centrifuge and discard the supernatant.

After adding to 4000, the mixture was stirred at 36 ° C for 3 days to inactivate HAV infectivity. SDS was added to the inactivated HAV extract, and the mixture was stirred at room temperature for 24 hours to obtain a 303-treated extract. SDS was added at various concentrations up to 1.2 wt%. 303 treatment 1 "1 extract contains 0.1% sodium azide, 5mM £ 0 and 0.9

It was diluted with 0.01M phosphate buffer (pH 7.2) containing% NaCl to obtain an SDS-treated HAV antigen reagent. SDS-treated HAV antigen reagents were stored at 4 ° C until use. Atsushi

In the same manner as in Example 1, the HAVAB-M positive panel sample was diluted with physiological saline. The diluted sample was further diluted 5-fold with IgM reagent (30〃1). As a comparison, the HAVAB-M negative panel sample was diluted with physiological saline, and further diluted 5-fold with the buffer used for diluting IgM. The diluted sample (125/1) was placed in a container, and an anti-human-IgM antibody-coated fine particle reagent (30/1) was added thereto, followed by reaction at 37 ° C for 20 minutes. The reacted microparticles were captured by a glass fiber filter and washed twice with 0.1 M borate buffer (pH 8.5) (300〃1). Next, SDS-treated HAV antigen reagent (30 / z 1) was added to the filter surface.

The reaction was carried out at 7 ° C for 20 minutes with the fine particles captured on one surface of the filter. The filters were washed once with 0.1 M borate buffer (pH 8.5) (100 / zl) and once with the same buffer (300 z1). Next, an acridinium-labeled anti-HAV antibody reagent (30 // 1) was added to the surface of the filter, and allowed to react at 37 ° C for 10 minutes with the fine particles captured on the surface of the filter. The filters were washed once with 0.1 M borate buffer (pH 8.5) (100〃1) and once with the same buffer (300 / z 1). The filter was transferred to a chemiluminescence reader, in which a trigger solution (5 μl) containing 0.4% hydrogen peroxide in 0.25N NaOH was fed to the filter. Ataridium bonded to the microparticles emitted light, and the amount of generated light was measured. The results obtained are shown in FIG. The horizontal axis in Fig. 1 is the concentration of the sample when the sample was diluted with physiological saline (first dilution). From FIG. 1, it was found that excellent dilution linearity was obtained by dilution with a solution containing human IgM. In this way, IgM is present in large amounts in serum, and even if the sample serum is diluted with a large amount of diluent, the amount of carrier-conjugated antibody used is not sufficient relative to the amount of IgM (for example, HAV infection). Person In the case of infection, the amount of IgM in blood increases several times to about ten-fold during infection), and the carrier-bound antibody can only bind to a part of blood IgM of total IgM. Thus, the amount of anti-HAV-IgM antibody (hereinafter simply referred to as “HAV-M”) that can bind to the carrier-bound antibody is eventually proportional to the amount of HAV-M and the amount of total IgM in blood. I understand. In other words, no matter how much the sample is diluted or concentrated, the amount of HAV-M bound to the carrier is always constant, and the amount of HAV-M bound to the carrier when the sample is diluted with a normal diluent. Turned out to be less. On the other hand, when the sample is diluted with a solution containing human IgM, the amount of HAV-M that can bind to the carrier-bound antibody is proportional to 118.1 \ amount / (total blood IgM amount + added IgM amount). The amount of HAV-M that can bind to the carrier-bound antibody can be adjusted according to the amount of added IgM regardless of the amount of total IgM in blood. In this way, it is possible to assemble a measurement system in which the measured HAV-M amount has dilution linearity regardless of the IgM amount in the sample. If qualitative measurement is sufficient, do not add IgM, or if you want to quantify,

By adding IgM, the linearity of the measured HAV-M amount and the linearity of the detected amount depending on the HAV-M concentration can be easily achieved. The invention's effect

By diluting the test sample with at least IgM or an aqueous solution containing IgM, a necessary measurement range can be obtained in the measurement of IgM antibodies in the sample, and more excellent measurement can be performed. Furthermore, it has become possible to assemble a wide range of automated measurement systems, avoiding restrictions on the amount of diluent. Detects specific IgM earlier in the phase (acute phase), such as HAV-related antibodies, or reduces the pre-dilution dilution to allow for a wider range of measurements, such as in cases of liver disease such as type A liver disease Diagnosis · Detection can be performed reliably.

Claims

The scope of the claims

1. In a method of immunologically measuring a specific IgM antibody to a target antigen in a test sample using an anti-human IgM antibody reagent, the test sample is analyzed using at least IgM or IgM. A method for measuring a specific IgM antibody, characterized by dilution with an aqueous solution containing the antibody.

2. The method for measuring a specific IgM antibody according to claim 1, wherein the specific IgM antibody is an IgM antibody at an early stage of infection.

3. The method for measuring a specific IgM antibody according to claim 1, wherein the specific IgM antibody is an IgM antibody against HAV or an IgM antibody against HBe in the early stage of infection.

4. The method for measuring a specific IgM antibody according to any one of claims 1 to 3, wherein the sample to be measured is whole blood, serum, or plasma.

5. (1) Dilute the sample to be measured with a buffer, diluent, or an aqueous solution of the diluent as necessary, and then dilute the sample with at least IgM or an aqueous solution containing IgM. Anti-human IgM

The IgM antibody in the sample is reacted immunologically with the solid-phase anti-human IgM antibody by reacting with the M antibody-bound solid carrier. (2) (a) A specific antigen reagent is added to the obtained reaction product. An immunological reaction is performed, and an antibody against the antigen reagent is labeled with a labeling agent.The labeled antibody is immunologically reacted, or (b) a specific antigen reagent is labeled with a labeling agent on the obtained reaction product. The method for measuring a specific IgM antibody according to any one of claims 1 to 4, wherein the method comprises immunologically reacting a labeled antigen.

6. The method for measuring a specific IgM antibody according to claim 5, wherein the labeled antibody is an acridinium-labeled anti-HAV antibody or an acridinium-labeled anti-HBc antibody.

7. The method for measuring a specific IgM antibody according to any one of claims 1 to 6, wherein the target antigen is a HAV antigen or an HBc antigen.

8. The method for measuring a specific IgM antibody according to claim 1, wherein the HAV antigen reagent is obtained by treating a HAV antigen obtained by an in vitro cell culture method with a surfactant.

9. The method for measuring a specific IgM antibody according to claim 8, wherein the surfactant is an anionic surfactant.

10. The method for measuring a specific IgM antibody according to claim 9, wherein the anionic surfactant is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS).