JP3487642B2 - Method for measuring HAV antigen and antibodies immunologically reactive with the antigen - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to hepatitis A virus (H
AV) An improved method for preparing an antigen, and more particularly, a method for preparing a hepatitis A virus antigen using a surfactant, particularly an anionic surfactant. Thus, there is provided a measurement reagent having improved measurement sensitivity when using a hepatitis A virus antigen, which is used in a method for measuring an antibody immunologically reactive with the antigen in a sample. The present invention also relates to an immunological assay for hepatitis A infection using a further improved hepatitis A virus antigen.

[0002]

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

By the way, although the incidence of hepatitis A virus infection is decreasing in Japan at present, the number of people who are negative for anti-HAV antibody is increasing mainly in the younger age group, while on the other hand, the number is higher in older people. 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. In addition, since HAV uses oral infection by feces as its main transmission route, the risk of infection in areas with poor environmental hygiene is great, and the number of overseas travel opportunities has increased recently. It is also emphasized in terms of preventing infections between relatives and employees.

In order to detect this HAV infection, HAV is used.
Anti-HA caused by strong immune reaction in vivo with infection
It is performed by detecting V antibody, and HAV antigen immunologically reactive with this anti-HAV antibody is used as a reagent. This HAV antigen is used as a reagent in an immunological assay method as it is, or after immobilizing or labeling HAV obtained from cultured cells infected with HAV as described above.

However, this H
The test method for detecting an antibody against AV is still problematic in the diagnosis of acute phase, its detection sensitivity, and its specificity are not always sufficient, and non-specific reaction is observed. Therefore, it is required to develop a measurement method having higher sensitivity and specificity. Detection of HAV-related antibodies at an earlier stage is important not only for promoting effective treatment but also for ensuring safety in the medical field, and it is also required to increase the detection rate in type A liver disease. ing. Up to now, cultured cells infected with HAV have been cultured for about 2 to 10 days, preferably 7 days, after removing the growth medium from the culture, the cells are lysed and the cell lysates thus obtained are used to The hepatitis A virus is obtained in this way by removing the substances of origin, cell organelles, crushed substances and the like by centrifugation.

By the way, in the lysis treatment of the infected cultured cells, the use of a non-physiological solution containing a chelating agent and a nonionic surfactant in a buffered solution may significantly increase the yield of the virus. However, the virus obtained from the cell lysate obtained by lysing the culture cells infected with HAV as described above is simply extracted as it is from a known formalin treatment, for example, infected cells. Incubation of the resulting virus at 37 ° C in a 1: 4000 dilution of a 37% formalin solution to inactivate its infectivity to form a HAV antigen, which is then used as an immunoassay to detect antibodies to HAV. Used as HAV antigen when performing. However, when such an HAV antigen is used, there is a problem in terms of sensitivity and it has been a factor of false negatives. As described above, it has been known so far to use a surfactant when lysing infected cells, but HAV once separated from the cell lysate obtained by lysis.
Was merely inactivated as it was and used as the HAV antigen. As described above, the low levels of HAV-related antibodies detected earlier, the detection rate in type A liver disease increased, and the low levels of HAV antigens used when diagnosing and detecting HAV infection, etc. There is an urgent and strong demand to reduce false negatives in the concentration range.

[0007]

[Means for Solving the Problems] The present inventors have found that an antibody immunologically reactive with a HAV antigen (HAV antibody) is more specific by using a viral antigen obtained by an in vitro cell culture method. As a result of earnest research to find a highly accurate and accurate measurement method, a method and a reagent capable of increasing reproducibility and higher sensitivity by a simple method were successfully developed.

The present inventors lysed infected cells when using a viral antigen obtained by an in vitro cell culture method to measure an antibody immunologically reactive with the antigen in a sample. HA once separated from the cell lysate obtained by
The V extract further treated with a surfactant was treated with HA
When used as a V-antigen reagent, unexpectedly little adverse effect was exerted on the antigen activity, and non-specific adsorption of the antigen could be suppressed. Furthermore, it has become possible to increase the amount of the HAV antigen reagent used in the measurement system. In this way, they have found that the measurement sensitivity can be significantly increased, and completed the present invention.

The present invention provides a HAV antigen reagent obtained by treating a HAV extract obtained from a cell lysate obtained by an in vitro cell culture method with at least a surfactant, particularly an anionic surfactant. provide. Furthermore, the present invention provides, in a reagent for measuring an antibody immunologically reactive with HAV, a HAV extract obtained from a cell lysate obtained by an in vitro cell culture method, at least a surfactant, particularly an anionic agent. HAV treated with a surfactant
Provided are a HAV antibody measuring reagent characterized by being used as an antigen reagent, and a measuring method using the reagent.

The HAV extract used in the present invention includes, for example, African green monkey kidney cultured cells, human liver tumor cell line PLC / PRF / 5, Hep. HAV-infected cells such as G2, which can be cultivated, and more preferably cell line cells capable of producing HAV in a large amount, 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, optionally with 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. 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 treated by a known method or a modified method thereof to inactivate its infectivity. 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 )liquid,
Piperazine-N, N'-bis (2-ethanesulfonic acid)
(PIBES) solution, 3- (cyanohexylamino) -1
-Propane sulfonic acid (CAPS) liquid, 3- (morpholino) propane sulfonic acid (MOPS) liquid, amino acid liquid and the like can be mentioned. 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 invention, the infectiously inactivated HAV extract thus obtained is then treated with a surfactant. Anionic surfactants are particularly suitable as the surfactant. As the anionic surfactant,
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, organic base salts such as triethanolamine of higher fatty acids having 12 to 18 carbon atoms, lauryl Sodium sulfate
Thorium (LDS), sodium dodecyl sulfate (SD
S) of which a higher fatty acid or sulfuric esters of higher alcohols having a carbon number of 12 to 18, alkyl sulfonates, alkyl aryl sulfonates such as sodium alkylbenzene sulfonate, and the like, in particular LDS, SDS shows remarkable effects. Examples of the alkali metal include sodium, potassium and lithium, and examples of the alkaline earth metal include calcium and magnesium.

The amount of these surfactants 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 treating the HAV extract 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 solution, 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 the resulting HA-treated extract.
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 aspect, the present invention comprises:
About 0.5% v / v to about 1.0% of the HAV extract obtained from the cell lysate obtained by the in vitro cell culture method.
Sodium dodecyl sulfate (SD at concentrations in the range of% v / v)
S) is mixed with the solution, and then, if necessary, for example, incubated at room temperature for 3 hours to obtain an SDS-treated HAV extract. In the present invention, HA in a sample characterized by using the obtained SDS-treated HAV extract
An immunoassay reagent for V antibody and a method for measuring an antibody in a sample using the same are provided.

In the present invention, when measuring an antibody immunologically reactive with an antigen in a sample, a radioimmunoassay, an enzyme immunoassay, a fluorescent immunoassay, a chemiluminescent immunoassay, or a fine particle labeling agent is used. Immunoassay and agglutination immunoassay.
In particular, during the acute phase of HAV infection, it is known that IgM type anti-HAV antibody appears in the blood of patients.
Acute hepatitis A infection can be diagnosed by measuring the anti-HAV antibody of the type. In this system for specifically measuring an IgM type anti-HAV antibody, a solid phase carrier coated with an anti-human IgM antibody is reacted with a measurement sample, and then (1) a labeled HAV antigen labeled with a labeling agent is reacted. Or (2) HAV
It is carried out by sequentially reacting with an antigen reagent and finally with a labeled anti-HAV antibody.

Therefore, in a more specific embodiment, the present invention comprises reacting the HAV antibody in the sample with an anti-human IgM antibody-bound solid support or a particulate support, etc., and reacting the HAV antibody in the sample with a solid-phase anti-human IgM antibody. Immunoreacting with the antibody, and then the surfactant-treated HAV extract obtained as described above, for example, SDS-treated HAV extract, is immunologically reacted, and the obtained reaction product is chemiluminescent. Provided is a method for measuring a HAV antibody, which comprises reacting a labeled anti-HAV antibody immunologically, and a reagent used in the method.

In the present invention, when measuring an antibody immunologically reactive with an antigen in a sample, the antigen or antibody involved in the antigen-antibody reaction may be, for example, agar, if necessary.
Agarose, cellulose, paper, nitrocellulose, dextran, gelatin, chitin, collagen, cotton and other bio-derived or natural product-derived polymers, polystyrene, polyethylene, polyvinyl alcohol, acrylic resins such as polyacrylamide, ion exchange resins, light Fine particles, beads, microplates, microtiter wells, microtubes, trips, membranes made of crosslinked resins, synthetic polymers such as Teflon and polyacetal, glass beads, silica gel, alumina, ceramics, carbon, inorganic materials such as magnesium sulfate, etc. , Trays, gels, etc., and further fixed on a solid carrier such as red blood cells, latex particles, emulsion, etc., and this solid carrier is brought into contact with a sample containing an antibody or the like to be analyzed, and thus solidified on the solid carrier. And antigens, specifically allowed binding reaction with an antibody or the like of the analysis sample, can be performed by detecting an analyte which is the specifically bound. Of course, it is preferable to use these solid carriers so as not to cause an unfavorable reaction with the surfactant used for treating the HAV antigen.

In a preferred embodiment, as the anti-human IgM antibody-bound solid carrier or particulate carrier to be reacted with the sample in the present invention, polystyrene beads,
Fine particles made of polystyrene or the like can be used.
As the antibody, an antibody against human IgM and HA
Any antibody can be used without particular limitation as long as it is an antibody against a target antigen such as an antibody against V. Antibodies can be obtained by a conventional method, for example, Shigeru Muramatsu, et al., Experimental Biology Course 14, Immunobiology, Maruzen Co., Ltd., 1985, Japan Biochemical Society, Sequel Chemistry Laboratory Course 5, Immunobiochemistry. Research method, Tokyo Kagaku Dojin, 1986, Japan Biochemical Society, Shinsei Chemistry Laboratory 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 1992, etc. , Bovine, sheep, rabbit, goat, rat, mouse or the like, or may be a monoclonal antibody. These may be used alone or in combination. 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 are 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.

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 analytical sample, a complex or the like, 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, a chemiluminescent immunoassay using a secondary antibody reagent labeled with acridinium ester or the like, or a fluorescent immunoassay using a secondary antibody reagent labeled with luminescent lanthanide, etc. The measuring method is a preferable method because it allows automated measurement. In particular, chemiluminescence immunoassay using a secondary antibody reagent labeled with acridinium ester is preferable because automated measurement can be performed.

Examples of the acridinium esters are, for example, JP-A-62-39598 and JP-A-62-619.
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.

In particular, Japanese Patent Laid-Open No. 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.

As the light emitting lanthanide, 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 the measuring method utilizing the agglutination reaction, a soluble antigen is generally bound to a particulate carrier, for example, so-called sensitized particle antigen in which erythrocytes, polystyrene particles, latex particles, etc. are bound, and an antibody thereto. It is possible to use a reaction in which and specifically bind to form an observable aggregate.

In order to bind or adsorb the solid carrier, the particulate carrier, the label or the like and the antigen or the antibody, a method generally used in the art can be used, and examples thereof include ionic interaction, hydrophobic interaction and covalent interaction. It can be performed by physical adsorption such as bonding or chemical bonding.
For example, as the cross-linking agent, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-o-phenylenedimaleimide, N-succinimidyl 3- (2-pyridyldithio) propionate, N -Succinimidyl S-acetylmercaptoacetate, 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) acetate, N-succinimidyl 4- (p-maleimidophenyl) 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 is a competitive assay,
Sandwich assay, neutralization assay, solid phase assay,
It can also be performed in a manner suitable for a chromatogram assay or the like. 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. These measurement target samples are
If necessary, it may be concentrated or diluted before use, but it is usually preferable to use it after diluting it with the above-mentioned diluent or the like. Further, the above-mentioned buffer, diluent, chelating agent, preservative and the like can be added and used. The sample may optionally be diluted 50-500 fold.

If necessary, the sample can be diluted with an aqueous solution of hepatitis-negative human serum, plasma or immunoglobulin-containing fraction.

Furthermore, the sample should be replaced with the above dilution if necessary, and the human IgM-containing fraction and purified human Ig
It was added to M of which aqueous solution can be to be measured without any purpose anti HAV-IgM antibodies being affected by the IgM content of the sample. In the case of an aqueous solution containing approximately 0.1% human IgM, the sample is approximately 50-200 in the aqueous solution.
You may dilute twice. It becomes possible to easily set the measurement sample, such as the adjustment of the sample concentration, and the application in the automated immunoassay system.

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. As described above, all of the suitable labeling agents known in the art suitable for automation and labeled can be used in the present invention as long as they meet the purpose.

In the measuring system of the present invention, HAV
Another surfactant other than the one used to process the antigen,
It may be used by containing a buffer, a diluent or diluent, a blocking agent, a chelating agent, a preservative and the like. Preferred other surfactants include polyoxyethylene sorbitan (representative is available under trade names such as Tween 20), polyoxyethylene ether (representative is such as Triton X-100). Available under the trade name), octylphenol-ethylene oxide condensate (typically Nonidet P-4
(Available under trade names such as 0).

Examples of the buffer, diluent or diluent include water, phosphate buffer, Tris buffer such as physiological saline, HEPES solution, PBES solution and CAPS as described above.
Liquid, MOPS liquid, amino acid liquid and the like. These may be used alone or in any combination. Examples of chelating agents include EDTA and EGTA. Examples of the preservative include sodium azide, ethylparaben and the like. In addition, in the assay system of the present invention, various animal sera such as bovine serum, bovine serum albunmin (BSA), fetal bovine serum (FCS), goat serum, albumen albunmin, gelatin, various milk proteins such as skim milk, casein, Those selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone and the like such as casein degradation products and whey proteins can be added.

[0037]

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 Anti-Human μ-IgM Antibody-Coated Microparticles Polyclonal antibody having specificity for human IgM μ chain obtained from goat (manufactured by Jackson Immuno Research Laboratories, USA [Jackson Immuno Re
search 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 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
100 μg / ml) at about 4 ° C. and 1% sodium caseinate, 0.1% Tween 20, 0.1% sodium azide, 5 mM EDTA and 0.9% NaC before use.
0.05M phosphate buffer containing 1 (pH 6.3)
Was diluted with to obtain an acridinium-labeled anti-HAV antibody reagent.

Preparation of SDS-treated HAV antigen HAV was prepared from Bas-Alexander cells (B
It culture | cultivated using arts-AlexanderCells. Add 0.5% Triton to the cultured cells.
n) containing X-100 and 5 mM EDTA and 0.
0.02M phosphate buffer containing 9% NaCl (pH 7.
After mixing 2) and stirring at 36 ° C. for 16 to 68 hours, centrifugation is performed, and the supernatant is discarded to add the HAV extract at a formaldehyde dilution ratio of 1: 4000, and then at 36 ° C. The HAV infectivity was inactivated by stirring for 3 days. SDS on inactivated HAV extract
Was added, stirred at room temperature for 24 hours, and then SDS-treated HAV
An extract was obtained. SDS was added at various concentrations up to 1.2 wt%. The SDS-treated HAV extract is
0.01 M phosphate buffer containing 0.1% sodium azide, 5 mM EDTA and 0.9% NaCl (pH
It was diluted with 7.2) and used as an SDS-treated HAV antigen reagent. SDS-treated HAV antigen reagent was stored at 4 ° C until use.

Assay HAVAB-M positive panel samples and HAVAB-M negative panel samples were diluted 201-fold with saline. The diluted sample (25 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, 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. The filters were washed once with 0.1 M borate buffer (pH 8.5) (100 μl) 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 captured on the filter surface at 37 ° C. for 10 minutes. 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 chemiluminescent 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 microparticles emitted light and the amount of light generated was measured. The obtained results are shown in FIGS. 1 and 2. The horizontal axis in FIG. 1 represents the SDS concentration in the SDS-treated antigen reagent. In FIG. 2, the amount of luminescence of the HAVAB-M positive panel sample is shown as HAVAB-
The relationship between the ratio divided by the luminescence amount of the M-negative panel sample and the SDS concentration is shown. From FIG. 2, it can be seen that by treating HAV with an appropriate amount of SDS, the ratio of signal (emission amount of positive panel sample) to noise (emission amount of positive panel sample) was significantly increased, and sensitivity was significantly improved. .

[0042]

By using a HAV antigen treated with a surfactant, particularly an anionic surfactant, as an antigen,
It becomes possible to set up a highly sensitive measurement system in the measurement of an antibody immunologically reactive with the antigen in a sample.
It is possible to provide an antibody measurement system that is immunologically reactive with a better HAV antigen, has high utility in clinical tests, and increases the detection rate.

[Brief description of drawings]

FIG. 1 shows the relationship between the SDS concentration in an SDS-treated antigen reagent and the luminescence amount of a sample.

FIG. 2 shows the relationship between the luminescence amount of the positive panel sample / the luminescence amount of the negative panel sample and the SDS concentration in the SDS-treated antigen reagent.

Claims (10)

(57) [Claims] 1. HAV produced in HAV-infected cultured cells
HAV characterized by using a HAV antigen treated with a surfactant in a method for measuring an HAV antibody using an antigen
Antibody measurement method. 2. HAV produced in HAV-infected cultured cells
The method for measuring HAV antibody using an antigen, wherein the HAV extract harvested from the HAV-infected cultured cells is used as a HAV antigen after being treated with at least a surfactant. . 3. The method for measuring HAV antibody according to claim 1 or 2, wherein the HAV antibody is human IgM. 4. The method for measuring a HAV antibody according to claim 1, wherein the surfactant is an anionic surfactant. 5. The method for measuring HAV antibody according to claim 4, wherein the anionic surfactant is sodium dodecyl sulfate (SDS ) . 6. The HAV extract lyses HAV-infected cultured cells, and HAV obtained from the resulting cell lysate.
The method for measuring an HAV antibody according to any one of claims 2 to 5, which is obtained by inactivating HAV infectivity. 7. The HAV antibody in the sample is reacted with an anti-human IgM antibody-bound solid support to immunologically react the HAV antibody in the sample with the solid-phase anti-human IgM antibody, and then harvested from HAV-infected cultured cells. HAV obtained by treating the extracted HAV extract with at least (a) an anionic surfactant
Immunoreacting with an antigen, the resulting reaction product is immunologically reacted with a labeled anti-HAV antibody, or (b) a HAV antigen obtained by treating with an anionic surfactant is a labeling agent. The method for measuring HAV antibody according to any one of claims 1 to 6, which comprises immunologically reacting a labeled antigen obtained by labeling with. 8. The method for measuring an HAV antibody according to claim 1, wherein the chemiluminescent label is an acridinium label. 9. HA harvested from HAV-infected culture cells
HAV antigen obtained by treating V extract with at least an anionic surfactant. 10. The HAV antigen according to claim 9, wherein the anionic surfactant is sodium dodecyl sulfate (SDS ) .