JPH085633A - Measuring method of specific igm antibody and its reagent - Google Patents

Abstract

PURPOSE:To obtain a positive diagnostic detection method by detecting a specific IgM, e.g. an antibody related to HAV(hepatitis A virus), or reducing the dilution ratio of predilution thereby realizing the measurement over a wide range and enhancing the detection rate of hepatitis A virus disease. CONSTITUTION:A sample is diluted, as required, by a butter, a dilution liquid or an aqueous solution and then it is further diluted by at least IgM or an aqueous solution containing IgM. S specific IgM antibody in the sample is then caused to react on a solid carrier bonded with an antihuman IgM antibody and the IgM antibody in the sample is caused to react immunologically on a solid phase antihuman IgM antibody to obtain a reaction produce. The reaction product is then caused to react immunologically on a specific antigen reagent and further caused to react immunologically on a labeled antibody for an antigen reagent or an antigen produced by labeling the reaction product with a specific antigen reagent thus measuring a specific IgM antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention uses an anti-human IgM antibody reagent to produce a specific Ig for a target antigen in a test sample.
In a method for immunologically measuring M antibody, the present invention relates to a specific IgM antibody measuring reagent characterized by diluting a test sample with at least IgM or an aqueous solution containing IgM, and a measuring method using the reagent.

[0002]

2. Description of the Related Art Immunological assay methods are widely used in clinical tests and diagnosis of diseases in humans.
It is also applied to animals in the fields of clinical examination, disease diagnosis, and analysis, measurement, quantification, and detection of a wide range of other measurement objects. This immunological assay method utilizes an antigen-antibody reaction between an antigen and an antibody against the antigen, but usually, the detection of occurrence of this antigen-antibody reaction can be visually discriminated or Alternatively, since it is difficult to detect, various methods have been developed to facilitate the detection and detection.

[0003] Of these, measuring the specific IgM antibody produced by the immune reaction in the living body particularly in the acute phase is noted because it is useful for diagnosing the initial infection such as viral infection and pathogen infection. ing. However, there is a problem that the specific IgM antibody is difficult to measure because it is interfered by rheumatoid factors and the like and IgG and the like. As a typical immunological assay method using this IgM measurement, an IgM antibody capture assay method can be mentioned, and examples thereof include diagnosis of hepatitis A virus (HAV) infection, and hepatitis B virus core. Antigen (Hepatit
is B virus core antigen: H
Bc), rubella, measles, mumps, etc.

In particular, hepatitis A virus (HAV) is 1
It was discovered by Feinstone et al. In 973 among the fecal materials of acute hepatitis A patients.
Since the proliferation of experimentally infected chimpanzee liver tissue was reported in 1977, and the proliferation in primary marmoset hepatocytes and rhesus monkey fetal kidney cells was reported in 1979, HAV was first established and established in cultured cell lines. It has been reported to grow in African green monkey kidney cells, human diploid cells and the like.

By the way, although the incidence of hepatitis A virus infection is currently decreasing in Japan, the number of people who are negative for anti-HAV antibody is increasing mainly in the younger age group. From the situation that many positive people are distributed,
Adults are more likely to come into contact with foods and drinks contaminated with HAV. In addition, once infected, adults are more likely to develop hepatitis. There is a need to detect the presence or absence of HAV infection.

[0006] In addition, since HAV uses oral infection by feces as its main transmission route, the risk of infection in areas where environmental hygiene is inadequate is large, and the number of overseas travel opportunities has increased recently. Inspection is also important in the sense of preventing infection between relatives and employees.

In order to detect HAV infection in this acute phase, an anti-HAV antibody, especially an IgM antibody specific to HAV, which appears in the blood of a patient due to a strong immune reaction in the body associated with HAV infection, is detected. This anti-HAV
The following IgM antibody capture assay using a HAV antigen immunologically reactive with the (IgM) antibody as a reagent has been developed. A method for diagnosing an infection of acute hepatitis A according to a typical IgM antibody capture assay uses an anti-human IgM antibody having specificity for the μ-chain of an IgM antibody, and the anti-human IgM antibody (μ-chain specific antibody ) Is coated with a solid phase, for example, a solid phase carrier as shown below, to react with a measurement sample, then with a HAV antigen reagent, and finally with a labeled anti-HAV antibody, and so on. Has been done.

However, in such an acute phase, anti-HAV (IgM) in a test sample such as blood, serum, plasma, etc.
The concentrations of specific IgM and total IgM to be measured, such as antibodies, vary. Generally, it is known that the total amount of IgM in blood is usually about 0.4 to 2 mg / ml, but the amount of antibody in the solid phase coated with the anti-human IgM antibody in the first reaction described above. However, there is a problem that it is necessary to pre-dilute the test sample, for example, high-dilution pre-dilution. Conventionally, the test sample has been prediluted with a buffer aqueous solution, a physiological saline solution, or the like. This reduces the amount of total IgM antibody, but cannot reduce the ratio of the specific IgM amount to the total IgM antibody amount. Therefore, there is a problem that it is difficult to obtain a necessary measurement range. Further, in the automated measurement system, there is a problem that the amount of diluting liquid is limited, and there is a problem that the measurement range is limited. As described above, specific IgM, for example, an HAV-related antibody can be detected at an earlier stage, or the dilution ratio of predilution can be reduced to enable a wider range of measurement, and for example, a high concentration region such as type A liver disease. There is an urgent and urgent need for reliable measurement methods in.

[0009]

The inventors of the present invention can easily set the necessary measurement range of the test sample which does not have the above-mentioned problems, and can detect a specific IgM such as HA at an earlier stage.
As a result of diligent research to detect a V-related antibody and to find a measurement method suitable for an automated measurement system that enables a wider range of measurement by reducing the dilution ratio of pre-dilution, as a result of a simple method They have found that they can solve these problems and completed the present invention.

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 is treated with at least IgM or an IgM-containing aqueous solution. A specific IgM antibody measuring reagent characterized by diluting and a measuring method using the reagent are provided. Examples of the sample to be measured 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 the sample to be measured. In the present invention, a sample is added with an aqueous solution of human IgM-containing fraction, purified human IgM, or the like,
This makes it possible to measure an IgM antibody specific to the antigen of interest without being affected by the amount of IgM in the sample. About 0.0
In the case of an aqueous solution containing 05% human IgM, the sample is about 10 to 500 times, preferably about 50 to 2 times with the aqueous solution.
It may be diluted to 00 times, more preferably to about 70 to 150 times, and most preferably to about 80 to 120 times. Human I
By the addition treatment of gM, it is possible to achieve a wider range of measurement, and it becomes possible to easily perform the measurement sample preparation, for example, the measurement range setting such as the adjustment of the sample concentration. Easy to apply.

The sample to be measured can be used by adding a buffer, a diluent, a chelating agent, a preservative and the like, if necessary, before the above treatment. The sample may be 50 to
You may dilute 500 times. As a buffer, a diluent or a diluent, water, a phosphoric acid or a phosphate buffer, a tris (hydroxymethyl) aminomethane (Tris) buffer, for example, a sodium chloride solution such as physiological saline, N- (2- Hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) (HEPES) solution, piperazine-N, N'-bis (2-ethanesulfonic acid) (PIBES) solution, 3-
(Cyanohexylamino) -1-propanesulfonic acid (CAPS) solution, 3- (morpholino) propanesulfonic acid (MOPS) solution, N, N′-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES) ) Liquid, N
-Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) solution, N- (2-acetamide)
Examples include 2-aminoethanesulfonic acid (ACES) solution and amino acid solution. These may be used alone or in any combination. As a chelating agent, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether)
-N, N, N ', N'-tetraacetic acid (EGTA) and the like can be mentioned. These chelating agents are about 0.01 mM
It can be used in the range of about 20 mM.

The sample may be diluted with an aqueous solution containing hepatitis-negative human serum, plasma or human IgM, if necessary. In a more specific embodiment, the invention provides that the sample is once diluted with an aqueous solution, such as a buffer, diluent or diluent, and then diluted with at least IgM or an IgM-containing aqueous solution before Having specificity for a specific antigen such as anti-virus specific IgM antibody of
The gM antibody is reacted with a solid phase carrier or the like coated with an anti-human IgM antibody to immunologically react the IgM antibody in the sample with the solid phase anti-human IgM antibody, and then (a) a specific antigen such as a virus. A reagent is immunologically reacted, and the resulting reaction product is immunologically reacted with a labeled antibody such as a chemiluminescence-labeled antiviral antibody, or (b) a specific antigen reagent such as a virus is labeled with a labeling agent. Specific IgM characterized by comprising immunologically reacting with a labeled antigen which has been labeled
An antibody measuring method and a reagent used in the measuring method are provided. An example of a particularly preferred assay system is a method of diagnosing an acute hepatitis A infection by measuring the blood of a patient with an IgM anti-HAV antibody that occurs during the acute phase of HAV infection. In this system for specifically measuring an IgM type anti-HAV antibody, a solid phase coated with a μ-chain specific anti-human IgM antibody, for example, a solid phase carrier as shown below is reacted with a measurement sample, Are reacted with a HAV antigen reagent, and finally with a labeled anti-HAV antibody.

The HAV antigen reagent used in the system for specifically measuring the IgM type anti-HAV antibody according to the present invention is
The viral antigen obtained by the in vitro cell culture method is used. Examples thereof include a HAV extract isolated from a cell lysate obtained by lysing infected cells or a derivative derived from the HAV extract. The HAV extract can be obtained from, for example, African green monkey kidney cultured cells, human liver tumor cell line PLC / PRF / 5, Hep. HAV such as G2
Infected cells that can be cultivated, and more preferably cell line cells that can produce large amounts of HAV, are cultured in a known growth medium such as Eagle's minimum essential medium (Eagle's).
MEM), Dulbecco's minimum essential medium (Dulbecc
o's MEM), PRM1-1640 (Gibco
Inc.), Eagle's MEM), N- (2-hydroxyethyl) piperazine-N '-(2-ethanesulfonic acid) (HEPES) buffer-added Eagle MEM, phosphate buffered L-15-a medium, Hanks Liquid (Hanks'
If necessary, supplemented with fetal calf serum (FCS), antibiotics such as pencillin and treptomycin, yeast extract, bactopeptone, lactalbumin hydrolyzate, and other cell growth factors in a growth medium such as balanced salt solution). It is obtained in the following manner by culturing in the following, and then from the cell culture thus obtained.

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 chelate such as EDTA. Wash with agent added. The cells thus isolated and harvested are typically ED
Chelating agents such as TA and polyoxyethylene ethers (typically 0.5% Triton X-1
Buffered solution, such as phosphate containing a nonionic surfactant (eg, available under a trade name such as 00), eg 1 m
M EDTA and 0.5% Triton X-100
Lysis treatment with a phosphate buffered solution containing, or a solution buffered with phosphate containing deoxycholate, and the cell lysate thus obtained, if necessary,
For example, incubation treatment for about 10 to 15 minutes, followed by centrifugation treatment, for example, about 1,000 to 20,000 ×
The HAV extract can be obtained by centrifugation at g, preferably about 2,000 to 10,000 × g for about 5 to 60 minutes, preferably about 10 to 30 minutes. Thus, the hepatitis A virus is obtained by centrifuging and removing the nuclear-derived substance, cell organelle, crushed material, etc. from the cell lysate. The HAV extract can also be obtained, for example, as described in US Pat. No. 4,721,675. If necessary, the HAV extract can be further purified by, for example, a chloroform extraction method, an enzyme treatment method, a sucrose concentration gradient centrifugation method, or the like.

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 deactivation process is
For example, it is treated with formalin solution, for example, at about 37 ° C for about 2
About 1: 3000 to 1:70 of 5-45% formalin solution
It can be carried out by incubating under a dilution of 00, for example, under a dilution of 1: 4000, but other suitable methods can be selected from known methods and applied. This treatment can be performed, for example, for 2 weeks, and may be shorter or longer than that. If necessary, a buffer solution, a diluent or a diluent, a chelating agent, a preservative and the like can be added to the treatment liquid for this treatment.

The buffer, diluent or diluent may be water,
Phosphate buffer, tris (hydroxymethyl) aminomethane (Tris) buffer, sodium chloride solution such as physiological saline, N- (2-hydroxyethyl) piperazine-N ′
-(2-ethanesulfonic acid) (HEPES) solution, piperazine-N, N'-bis (2-ethanesulfonic acid) (PI
BES) liquid, 3- (cyanohexylamino) -1-propanesulfonic acid (CAPS) liquid, 3- (morpholino) propanesulfonic acid (MOPS) liquid, amino acid liquid and the like. These may be used alone or in any combination. As a chelating agent, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether) -N, N,
N ', N'-tetraacetic acid (EGTA) etc. are mentioned.

According to the present invention, the infectiously inactivated HAV extract thus obtained is used as it is for HA.
It is possible to use it as a V antigen, or to use it obtained by further treating it with a surfactant next, and such a surfactant-treated HAV extract can be used as a preferable one. As the surfactant, an appropriate surfactant can be selected from known or commercially available ones, and an anionic surfactant is particularly suitable.

Examples of the anionic surfactants include alkali metal salts or alkaline earth metal salts of higher fatty acids having 12 to 18 carbon atoms such as potassium stearate, alkali metal salts of bile acids, higher fatty acids having 12 to 18 carbon atoms. Organic base salts such as triethanolamine, sodium lauryl sulfate (LDS), sodium dodecyl sulfate (SDS) and other higher fatty acids having 12 to 18 carbon atoms or sulfuric acid esters of higher alcohols, alkyl sulfonates, sodium alkylbenzene sulfonates, etc. Alkylaryl sulfonates and the like can be mentioned, and particularly LDS and SDS show remarkable effects. Alkali metals include sodium, potassium and lithium, alkaline earth metals include calcium,
Examples include magnesium.

The amount of these surfactants to be used is preferably selected according to the amount of coexisting protein, and can be used, for example, in the range of about 0.001% v / v to about 10% v / v. . Particularly preferably, SDS is used, and about 0.05
% V / v to about 5% v / v, more preferably about 0.5% v / v to about 1.% in the absence of coexisting other proteins.
It can be used in the range of 0% v / v.

When the HAV extract is treated with a surfactant, the HAV extract is diluted with a buffer, a diluent or a diluent, if necessary, and mixed with a surfactant solution giving a required concentration, or suspended. It becomes cloudy. The mixture thus obtained can be stirred if necessary. Further, in some cases, glass beads or the like may be added to the mixture and stirred. As long as the stirring treatment can improve the measurement sensitivity, for example, only gentle mixing can be performed, or vigorous stirring and mixing can be performed. The treatment can be carried out at room temperature, under cooling, or at 37 ° C. or higher as long as it can improve the measurement sensitivity. The HAV extract treated with the surfactant can be used as it is for the next treatment, or it can be stored once and then used for the next treatment, and if necessary, subjected to a separation treatment such as centrifugation and the like. It can be used for the next treatment after being subjected to treatment such as washing. These treatments can be selected so as to suppress non-specific adsorption during measurement and improve sensitivity.

A buffer, a diluent or a diluent, a chelating agent, a preservative and the like can be added to the treating solution for treating the surfactant of the present invention. Examples of the buffer, diluent or diluent include water, phosphoric acid or phosphate buffer, Tris buffer, sodium chloride solution such as physiological saline, HEPES solution, PIBES solution, CAPS solution,
MOPS liquid, N, N'-bis (2-hydroxyethyl)
2-Aminoethanesulfonic acid (BES) solution, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES) solution, N- (2-acetamido) -2-
Examples thereof include aminoethanesulfonic acid (ACES) solution and amino acid solution. These may be used alone or in any combination. As a chelating agent,
Examples include EDTA and EGTA.

According to the present invention, the HAV extract is optionally treated with the above-mentioned surfactant prior to inactivating its infectivity, and then with the resulting surfactant-treated HA.
V 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, for example, about 37% formalin solution 1:37 at about 37 ° C.
It can be performed by performing an incubation treatment under 4000 dilution.

In a more specific embodiment, the HAV antigen reagent used in the present invention is a HAV extract obtained from a cell lysate obtained by an in vitro cell culture method at about 0.5% v / v. It is provided by mixing with a sodium dodecyl sulfate (SDS) solution having a concentration in the range of about 1.0% v / v, and then optionally incubating, for example, at room temperature for 3 hours to obtain an SDS-treated HAV extract. It can also be one. In the present invention, H in a sample using at least IgM or an IgM-containing aqueous solution treatment step and using the obtained SDS-treated HAV extract
An immunoassay reagent for AV antibody and a method for measuring an antibody in a sample using the same are also provided.

The specific IgM antibody in the present invention can be measured by methods such as radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, and chemiluminescence immunoassay. In particular, a chemiluminescence immunoassay method, for example, an IgM capture solid phase chemiluminescence immunoassay method is a preferable method because it can perform an automated measurement.

In the present invention, when measuring a specific IgM antibody in a sample, the antigen or antibody involved in the antigen-antibody reaction may be, for example, agar, agarose,
Bio-based polymers such as cellulose, paper, nitrocellulose, dextran, gelatin, chitin, collagen, and cotton, or polymers derived from natural products, acrylic resins such as polystyrene, polyethylene, polyvinyl alcohol, polyacrylamide, ion-exchange resins, photo-crosslinking resins , Synthetic polymers such as Teflon, polyacetal, glass beads,
Fine particles, beads, microplates, microtiter wells, microtubes, trips, membranes, trays, gels, etc. made of inorganic materials such as silica gel, alumina, ceramics, carbon, magnesium sulfate, etc., as well as red blood cells, latex particles, emulsions, etc. The solid carrier is fixed in advance, the fixed carrier is brought into contact with a sample containing an antibody or the like as an analysis target, and thus the antigen fixed on the fixed carrier and the antibody or the like in the analytical sample are specifically bound to each other. It can be carried out by reacting and detecting the specifically bound analyte.

In a preferred embodiment, polystyrene beads are used as the anti-human IgM antibody-bound solid carrier or particulate carrier to be reacted with the sample in the present invention.
Fine particles made of polystyrene or the like can be used.
Further, the antibody can be used without particular limitation as long as it is an antibody against human IgM. The antibody can be obtained by a conventional method, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985.
Ed., Biochemical Society of Japan, sequel to biochemistry experiment course 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 1986, ed.
Newborn Chemistry Experiment Course 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, immunize, for example, horses, cattle, sheep, rabbits, goats, rats, mice, etc. Alternatively, they may be obtained as a monoclonal antibody or may be monoclonal antibodies. These may be used alone or in combination with each other. If necessary, these antibodies may be digested with an enzyme such as pepsin or papain and used as F (ab ′) ₂ or Fab ′. The anti-human IgM antibody preferably includes an antibody that specifically reacts with the μ chain and an anti-μ chain-human IgM antibody, which were obtained by using a cell fusion technique using mouse myeloma cells. It goes without saying that it may be a monoclonal antibody. In addition, anti-HAV antibody
Those obtained from human HAV-positive serum or plasma having a high titer can be used, but needless to say, it may be a monoclonal antibody as described above. The anti-HBc antibody can be obtained from a virus culture, but can also be a recombinant antigen expressed in Escherichia coli, yeast, etc. using a gene recombination technique.

Radioimmunoassay, enzyme immunoassay,
For fluorescent immunoassay, chemiluminescent immunoassay, etc.,
Radioactive substances such as ¹²⁵ I and ³ H, horseradish peroxidase, β-D-galactosidase, enzymes such as alkaline phosphatase, fluorescent dyes such as fluorescein, chemiluminescent dyes such as acridinium esters, colloidal gold and selenium. An antigen or a secondary antibody labeled with a colored substance such as colloid or colored latex particles is used as a reagent, and specifically reacted with an antibody in an analysis sample, a complex, etc., and its radioactivity, enzyme activity, It is possible to determine whether or not the antibody or the like was present in the sample by measuring chemiluminescence or the presence or absence of color. In the present invention, in particular, a chemiluminescent labeling method, for example, an acridinium ester or a fluorescence labeling method, for example, a fluorescence or chemiluminescence immunoassay using a secondary antibody reagent labeled with a luminescent lanthanide or the like is an automated measurement. Is a preferable method. In particular, chemiluminescence immunoassay using a secondary antibody reagent labeled with acridinium ester is preferable because automated measurement can be performed.

As the acridinium esters, for example, JP-A-62-39598 and JP-A-62-619 are known.
69, JP-A-63-57572, JP-A-6
JP-A-3-101368, JP-A-63-112564, JP-A-1-199949, and JP-A-1-26.
1461, JP-A-2-96567, JP-A-2-133469, JP-A-2-503268, JP-A-2-501772, European Patent Publication No. 0082636, British Patent Specification No. 1 , 461, 8
77, U.S. Pat. No. 3,539,574 and the like N-alkyl or aryl acridinium-9-
Examples thereof include carboxylic acid esters.

Particularly, JP-A-63-112564,
1 described in US Pat. No. 3,539,574, etc.
0-alkyl N-alkyl or aryl-sulfonyl-N-alkyl or arylsulfonylacridinium-9-carboxamide, N-methylacridinium-9
-Carboxylic acid ester etc. are mentioned as a typical chemiluminescent label. In the case of acridinium labeling, treatment with a trigger reagent prior to measurement, eg hydrogen peroxide, eg about 0.01
% To about 0.1% hydrogen peroxide solution and sodium hydroxide, for example, about 0.05 N to about 0.5 N sodium hydroxide solution, and then the measurement can be performed using a luminometer or the like. it can.

Examples of the light emitting lanthanide include, for example, European Patent Publication No. 0068875 and US Pat.
74,120, U.S. Pat. No. 4,283,382.
U.S. Pat. No. 4,259,313, U.S. Pat. No. 4,352,751, U.S. Pat.
No. 432,907, European Patent Publication No. 0103558.
And the like, which can chelate a luminescent lanthanide having an aminocarboxylic acid group described in No. Further, the measurement can be facilitated by performing an excitation treatment with a laser beam or the like before the measurement. Of course, the labeling agent is not limited to the above-mentioned ones, and in consideration of the equipment used for measurement, the place, etc., the labeling agent is appropriately selected and used from those known to be used in the relevant field according to the purpose. be able to.

In order to bind or adsorb the solid carrier, the particulate carrier, the label or the like and the antigen or the antibody or the like, a method generally used in the art can be used, for example, ionic interaction, hydrophobic interaction and covalent interaction. It can be performed by physical adsorption such as bonding or chemical bonding.
For example, as a crosslinking agent, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-o-phenylenedimaleimide, N-succinimidyl 3- (2-pyridyldithio) propionate, N -Succinimidyl S-acetyl mercaptoacetate, N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, N-succinimidyl 6-maleimidohexanoate, N-succinimidyl 4-iodoacetylaminobenzoate, N-succinimidyl 3 -(P-hydroxyphenyl) propionate, N-succinimidyl m-maleimidobenzoate, N-succinimidyl 4-maleimidobutyrate, N-succinimidyl (p-maleimidophenyl) Seteto and N- succinimidyl 4-(p-maleimido phenyl) butyrate and the like.

Examples of solid carriers, particulate carriers, etc. are:
Examples include those mentioned above, for example, agar, agarose, crosslinked agarose, crosslinked alginic acid, crosslinked guar gum,
Cellulose ester such as nitrocellulose or carboxyl cellulose or mixed cellulose ester, gelatin, cross-linked gelatin, latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, Styrene-methacrylate copolymer, polyglycidyl methacrylate,
Polyesters such as acrolein-ethylene glycol dimethacrylate copolymers, natural or synthetic modified or unmodified polymerized carbohydrates such as polyamides, polyurethanes, polyepoxy resins, polymerized hydrocarbons, cross-linked derivatives thereof, glass, etc. Examples include materials selected from the group consisting of inorganic materials such as glass, silica gel, kaolin, talc, silica-alumina, alumina, and barium sulfate in the form of porous gel, fine particles, and the like. In the present invention, the measurement can be carried out in a manner suitable for competitive assay, sandwich assay, neutralization assay, solid phase assay, chromatogram assay and the like.

In the present invention, as the labeling reagent, 4
-Hydroxyphenylacetic acid, 1,2-phenylenediamine, tetramethylbenzidine and the like with horseradish peroxidase, umbelliferyl galactoside, nitrophenylgalactoside and the like with β-D-galactosidase,
Umbelliferyl phosphate, nitrophenyl phosphate, NADP etc. and alkaline phosphatase, enzyme reagents such as glucose-6-phosphate dehydrogenase, radioactive reagents, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, acridinium esters, Fluorescent reagents, luminescent reagents, chemiluminescent reagents, colloidal gold, which use luminescent lanthanides, etc.
Colloid labeling reagents such as silver colloid and selenium colloid,
A magnetic substance reagent, a hapten-labeled anti-hapten antibody detection system reagent such as biotin-labeled anti-biotin antibody, and the like can be used. As described above, in the present invention, the use of a chemiluminescent labeling reagent such as an acridinium ester or a fluorescent labeling reagent such as a secondary antibody reagent labeled with a luminescent lanthanide is particularly preferable because the measurement is automated.

In the measuring system of the present invention, surfactants, buffers, diluents or diluents other than those mentioned above, blocking agents, chelating agents, preservatives and the like can be added. As this preferable surfactant, polyoxyethylene sorbitan (representative one is
Available under trade names such as Tween 20), polyoxyethylene ether (typical is Triton)
X-100 and the like, and octylphenol / ethylene oxide condensates (typical ones are Nonidet P-40 and the like).

As the buffer, diluent or diluent, HEPES solution, PBES solution, CAPS solution, M, etc. as described above such as water, phosphate buffer solution, Tris buffer solution, physiological saline solution, etc.
Examples thereof include OPS liquid and amino acid liquid. These may be used alone or in any combination. Examples of chelating agents include EDTA and EGTA.

Examples of preservatives include sodium azide and ethylparaben. In addition, in the assay system of the present invention, various animal sera, such as bovine serum, bovine serum albunmin (BSA), fetal bovine serum (FC
S), goat serum, albumen albumin, gelatin, various milk proteins such as skim milk, casein, casein degradation product, whey protein, and the like, selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, and the like can be added.

In the present invention, the reagents may be contained in a single container or a plurality of containers, and may be blended for use. In a more specific embodiment of a typical measurement system for diagnosing HAV infection using IgM antibody measurement, the present invention comprises diluting a sample with an appropriate concentration of physiological saline or the like, for example, about 80 to 120 times, preferably Is diluted about 100 times, and then an appropriate amount of a human IgM solution and an anti-human IgM antibody-bound solid carrier or a particulate carrier are reacted, and then (1) a HAV extract harvested from HAV-infected cultured cells or (2) H obtained by treating this HAV extract with at least a surfactant such as SDS
The reaction product obtained by immunologically reacting with an AV antigen is immunologically reacted with a chemiluminescence-labeled anti-HAV antibody, for example, an acridinium-labeled anti-HAV antibody, and is a trigger comprising a hydrogen peroxide solution and a sodium hydroxide solution. A method for measuring an HAV antibody, which comprises detecting after reacting with a reagent, can be provided.

When a viral antigen obtained by an in vitro cell culture method is used to measure an antibody immunologically reactive with the antigen in a sample, a cell lysate obtained by lysing infected cells When the HAV extract once separated from the above is further treated with a surfactant and used as a HAV antigen reagent, the measurement sensitivity can be significantly increased without unexpectedly adversely affecting the antigen activity. .

In the present invention, it can be used for a known immunological assay for measuring a specific IgM antibody,
For example, it is considered that the method can be applied to a specific antibody measuring system generated by virus infection, pathogenic microbial infection and the like. Viruses include herpes simplex, herpes zoster, varicella virus, mumps, measles, rubella, AIDS virus (HI
V), hepatitis B virus (HBV), hepatitis C virus (HCV), and the like. Examples of pathogenic microorganisms include diphtheria (Corynebacteium diphtheriae), tetanus (Clostridium tetani), and cholera (Vibrio cho).
lerae), Clostridium botulinum (Clostridium botulinum),
Clostridium perfringens, Staphylococcus aureus, for example, MRSA, Shigella dysenteriae, Bordetella
perussis), Salmonella typhi, Salmonella paratyphi, and Salmonel
la enteritidis), Mycobacterium tuberculos
is) etc.

[0040]

EXAMPLES Next, the present invention will be described more specifically by showing examples, but it is understood that the present invention is not limited to these examples and can be carried out in various forms as long as the idea thereof is followed. Should be.

Example 1 Preparation of IgM A commercially available human IgM solution (manufactured by Chemicon International Inc. [Chemicon International]
al, USA. ]) Was diluted in 0.02 M Tris buffer (pH 8.5) containing 0.1% sodium azide before use to give an IgM reagent.

Preparation of Anti-Human μ-IgM Antibody-Coated Microparticles A polyclonal antibody (Jackson ImmunoRese manufactured by Jackson ImmunoResearch Laboratories, Inc., USA) also has specificity for human IgM μ chains obtained from goats.
arch Labo. , USA. ]) Was bound to carboxylated polystyrene latex fine particles ([Seradyn, USA.]; 0.2 μm, manufactured by Celadaine, USA) by the method described below. First, 0.015M MES
1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDAC; 16 mg / in (2- [N-morpholino] ethanesulfonic acid) buffer (pH 4.7)
ml) was used to bind the polyclonal antibody anti-human IgM antibody (160 mg / ml) at room temperature for 1.5 hours. Then 1% Tween 20 and 0.9%
0.05M phosphate buffer containing NaCl (pH
Washed with 7.2). Finally, in 0.01 M Tris buffer (pH 7.4) containing 0.05% gelatin, 0.1% Tween 20, 0.9% NaCl and 0.1% sodium azide. Stored in. It was diluted with a storage buffer so that the solid content of the coated microparticles was 0.0625%, and this was used as an anti-human μ-IgM antibody-coated microparticle reagent.

Preparation of acridinium-labeled anti-HAV antibody β-alanine acridinium (1 mg) was dissolved in anhydrous dimethylformamide (DMF) (100 μl) and N 2 was added.
-Hydroxysuccinimide (NHS) (5.75 mg
/ Ml, 50 μl) and EDAC (9.6 mg / ml,
50 μl) was continuously added and activated by stirring at 25 ° C. in the dark for 48 hours. Activated acridinium was added to 0.1 M phosphate buffer (pH 8.0) containing 0.9% NaCl and 0.5% CHAPS containing purified protein A monoclonal anti-HAV antibody (1 mg / ml). (4 times the number of moles of antibody), the reaction mixture was stirred at room temperature for 10 minutes. Buffer 0.1% CHAP
After replacement with 0.01 M phosphate buffer (pH 6.3) containing S, 0.1% sodium azide and 0.9% NaCl, the preparation was centrifuged, and the supernatant was replaced. SEC 2 equilibrated with the same buffer as
50 (manufactured by BioRad, USA [Biolad, USA.]
Chromatography on an HPLC column. Each fraction (1 ml) was added at 369 nm and 28
The amount of bound acridinium was determined by analysis by UV spectroscopy at 0 nm. Concentrate the bound fraction (about 1
(00 μg / ml) at about 4 ° C. and before use 1% sodium caseinate, 0.1% Tween 20, 0.1% sodium azide, 5 mM EDTA and 0.9% NaC.
0.05M phosphate buffer containing 1 (pH 6.3)
Was diluted with to obtain an acridinium-labeled anti-HAV antibody reagent.

Preparation of HAV Antigen HAV was derived from Berth-Alexander cells (B
It culture | cultivated using arts-Alexander Cells. Add 0.5% Triton to the cultured cells.
on) X-100 containing 0.02M phosphate buffer (pH 5 mM EDTA and 0.9% NaCl)
7.2) were mixed and stirred at 36 ° C. for 16 to 68 hours, then centrifuged, and the supernatant was discarded to give HAV.
The extract was added to formaldehyde at a dilution ratio of 1: 4000 and then stirred at 36 ° C for 3 days to obtain HAV.
Inactivated the infectivity of. The inactivated HAV extract is 1% bovine serum albumin, 0.05% Tween 20,
0.1% sodium azide, 5 mM EDTA and 0.
0.01 M phosphate buffer containing 9% NaCl (p
It was diluted with H7.2) and used as the HAV antigen reagent. The HAV antigen reagent was stored at 4 ° C until use.

Assay HAVAB-M positive panel samples were diluted with saline. The diluted sample was further diluted 5-fold with IgM reagent (30 μl). For comparison, a HAVAB-M negative panel sample was diluted with physiological saline and then further diluted 5-fold with the buffer used for diluting IgM. The diluted sample (125 μl) was placed in a container, and anti-human μ-
Add IgM antibody-coated microparticle reagent (30 μl) and
The reaction was carried out at 0 ° C for 20 minutes. The reacted fine particles are captured by a glass fiber filter, and 0.1M borate buffer solution (pH
It was washed twice with 8.5) (300 μl). Next, add the HAV antigen reagent (30 μl) to the filter surface and incubate at 37 ° C.
It was made to react with the fine particles captured on the filter surface for 20 minutes. Filter with 0.1 M borate buffer (pH
8.5) (100 μl) once and the same buffer (30 μl)
It was washed once with 0 μl). Next, acridinium labeled anti-H
Add AV antibody reagent (30 μl) to the filter surface,
The reaction was carried out at 37 ° C. for 10 minutes with the fine particles captured on the filter surface. The filters were washed once with 0.1 M borate buffer (pH 8.5) (100 μl) and once with the same buffer (300 μl).

The filter was transferred to a chemiluminescence reader in which a trigger solution (0.41) containing 0.4% hydrogen peroxide in 0.25N NaOH was pumped into the filter. Acridinium bound to the particles emits light,
The amount of light generated was measured. At low HAVAB-M positive panel sample concentrations, 21 with dilution with IgM reagent
36 luminescence (photon counts), 13299 luminescence without addition of IgM reagent (photon counts) and IgM at medium HAVAB-M positive panel sample concentrations.
With the dilution treatment with the reagent, the luminescence amount of 5010 (photon count) and without the addition of the IgM reagent were the luminescence amount (photon count) of 16788, and at the high HAVAB-M positive panel sample concentration, the dilution treatment with the IgM reagent was 12
The emission amount (photon count) was 531 and the emission amount (photon count) was 18029 without addition of the IgM reagent.
Incidentally, when the SDS-treated HAV antigen was used as in Example 2 below (IgM reagent added), it was 43 in the low value panel.
The light emission amount (photon count) was 81, the medium value panel was 11007, and the high value panel was 11007.

Example 2 Preparation of SDS-treated HAV Antigen HAV was prepared from Barth-Alexander cells (B
It culture | cultivated using arts-Alexander Cells. Add 0.5% Triton to the cultured cells.
on) X-100 containing 0.02M phosphate buffer (pH 5 mM EDTA and 0.9% NaCl)
7.2) were mixed and stirred at 36 ° C. for 16 to 68 hours, then centrifuged, and the supernatant was discarded to give HAV.
The extract was added to formaldehyde at a dilution ratio of 1: 4000 and then stirred at 36 ° C for 3 days to obtain HAV.
Inactivated the infectivity of. SDS was added to the inactivated HAV extract, and the mixture was stirred at room temperature for 24 hours to obtain an SDS-treated HAV extract. SDS was added at various concentrations up to 1.2 wt%. The SDS-treated HAV extract contained 0.1% sodium azide, 5 mM EDTA.
And a 0.01 M phosphate buffer (pH 7.2) containing 0.9% NaCl were used as SDS-treated HAV antigen reagents. SDS-treated HAV antigen reagent was stored at 4 ° C until use.

Assay As in Example 1, HAVAB-M positive panel samples were diluted with saline. Dilute the sample with IgM reagent (30
μl) and further diluted 5-fold. HA as a comparison
The VAB-M negative panel sample was diluted with physiological saline and then further diluted 5-fold with the buffer used for diluting IgM. The diluted sample (125 μl) was placed in a container, and the anti-human μ-IgM antibody-coated microparticle reagent (3
0 μl) was added and reacted at 37 ° C. for 20 minutes. Capture the reacted fine particles with a glass fiber filter and
It was washed twice with the borate buffer solution (pH 8.5) (300 μl). Next, SDS-treated HAV antigen reagent (30 μl) was added to the filter surface and reacted with the fine particles captured on the filter surface at 37 ° C. for 20 minutes. Filter with 0.1M borate buffer (pH 8.5) (100μ
1) and once with the same buffer (300 μl). Next, an acridinium-labeled anti-HAV antibody reagent (30
μl) was added to the filter surface and reacted with the fine particles trapped on the filter surface at 37 ° C. for 10 minutes. Filter the filter with 0.1 M borate buffer (pH 8.5) (1
00 μl) and once with the same buffer (300 μl)
Washed twice.

The filter was transferred to a chemiluminescence reader in which a trigger solution (0.4 μl) containing 0.4% hydrogen peroxide in 0.25 N NaOH was pumped into the filter. Acridinium bound to the particles emits light,
The amount of light generated was measured. The obtained results are shown in FIG.
The horizontal axis in FIG. 1 represents the concentration of the sample when the sample was diluted with physiological saline (first dilution), and the one that was not diluted at all was 1
And From FIG. 1, it was revealed that excellent dilution linearity can be obtained by diluting with a solution containing human IgM.

Thus, even if a large amount of IgM is present in the serum and the sample serum is diluted with a large amount of diluent, the amount of the carrier-bound antibody used is not sufficient for the amount of IgM (for example, for a person infected with HAV). In such a case, the amount of IgM in blood increases by several to several ten times at the time of infection), and the carrier-bound antibody can only bind to a part of blood IgM out of all IgM. Anti-HA that can bind with
V-IgM antibody (hereinafter, simply referred to as "HAV-M")
It was found that the amount of H.V. was eventually proportional to the amount of HAV-M / the total amount of IgM in blood. In other words, no matter how much the sample is diluted or how much the sample is concentrated, the amount of HAV-M bound to the carrier will always be constant, and the HAV-M bound to the carrier will be bound when the sample is diluted with an ordinary diluent.
It turns out that the quantity is only getting smaller. On the other hand, when the sample is diluted with a human IgM-containing solution, the amount of HAV-M that can bind to the carrier-bound antibody is HAV-M amount / (total IgM in blood).
(Total amount + added IgM amount)
Regardless of the amount of M, the amount of HAV-M that can bind to the carrier-bound antibody can be adjusted according to the amount of added IgM. In this way, it is possible to assemble a measurement system in which the measured HAV-M amount has a dilution linearity regardless of the IgM amount in the sample. If qualitative measurement can be performed in this way, measurement is performed without addition of IgM, or if quantification is desired, addition linearity of HAV-M amount measured by adding IgM, and HAV-M
It is possible to easily achieve linearity of the detection amount depending on the concentration.

[0051]

INDUSTRIAL APPLICABILITY At least IgM or Ig
By diluting with an M-containing aqueous solution, it is possible to obtain a necessary measurement range in the measurement of IgM antibody in a sample,
Better measurement is possible. Furthermore, it became possible to avoid the restriction of the amount of diluent and to assemble a wide range of automated measuring systems. Specific Ig in earlier period (acute period)
M, for example, an HAV-related antibody can be detected, or the dilution ratio of the pre-dilution can be reduced to enable a wider range of measurement, and for example, the diagnosis and detection of type A liver disease can be reliably performed.

[Brief description of drawings]

FIG. 1 shows the relationship between sample concentration and luminescence amount.

Claims (10)

[Claims] 1. 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 is at least IgM.
Alternatively, a method for measuring a specific IgM antibody, which comprises diluting with an IgM-containing aqueous solution. 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 HBc in the early stage of infection. 4. The specific IgM according to any one of claims 1 to 3, wherein the sample to be measured is whole blood, serum, or plasma.
Antibody measurement method. 5. (1) A sample to be measured is diluted with a buffer, a diluent or an aqueous solution of a diluent as required, and then the sample is diluted with at least IgM or an aqueous solution containing IgM, and then the sample to be measured is specific to the sample. Anti-human I
(2) by reacting with a solid carrier bound to a gM antibody to immunologically react the IgM antibody in the sample with a solid phase anti-human IgM antibody;
(A) a specific antigen reagent is immunologically reacted with the obtained reaction product, and a labeled antibody obtained by labeling an antibody against the antigen reagent with a labeling agent is immunologically reacted, or (b)
5. The specific Ig according to any one of claims 1 to 4, which comprises immunologically reacting a labeled antigen obtained by labeling a specific antigen reagent with a labeling agent with the obtained reaction product.
Method for measuring M antibody. 6. The labeled antibody is an acridinium-labeled anti-HA.
The method for measuring a specific IgM antibody according to claim 5, which is a V antibody or an acridinium-labeled anti-HBc antibody. 7. The method for measuring a specific IgM antibody according to claim 1, wherein the target antigen is a HAV antigen or an HBc antigen. 8. The specific IgM according to claim 1, wherein the HAV antigen reagent is a HAV antigen obtained by an in vitro cell culture method and treated with a surfactant.
Antibody measurement method. 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 sodium lauryl sulfate (LDS).