JPH09127114A - Stabilized igm reagent for immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an IgM reagent stable over a long term and useful as an immunoassay reagent. SOLUTION: Regarding an IgM reagent used in the immunoassay. lgM or IgM contained water solvation stabilized with bovine serum albumin liquid is used as an IgM reagent. Regarding the method for measuring a singular IgM antibody for an object antibody in a specimen in an immunological way, using an anti-human IgM antibody reagent, in particular, a reagent at least made of IgM contained water solution stabilized with bovine serum albumin liquid is used to dilute the specimen, thereby providing a more accurate singular IgM antibody measurement method. Also, regarding a measurment system using a marked IgM antibody obtained by marking the IgM antibody with a marker, the marker, IgM antibody solution is stabilized at least by use of bovine serum albumin liquid, thereby providing a more stable method for measurirng an antigen contained in a specimen.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention provides a stabilized IgM reagent useful as an immunological assay reagent. Especially Ig
When the M or IgM-containing aqueous solution is stabilized with bovine serum albumin solution, the stabilized IgM or IgM-containing aqueous solution uses an anti-human IgM antibody reagent to produce a specific IgM antibody against the target antigen in the test sample, such as hepatitis A. It is useful as a reagent in a method for immunological measurement in diagnosis of virus (Hepatitis A virus: HAV) infection and the like. Further, when an IgM reagent labeled with a labeling agent is used in an antigen measurement system by a so-called sandwich assay method, the present invention also relates to an immunological measurement method in which the labeled IgM reagent is stabilized with bovine serum albumin solution.

[0002]

2. Description of the Related Art Immunoassays are widely used in human clinical tests and disease diagnosis.
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. It is known that immunoglobulins, ie, antibodies, can be classified into isotype classes such as IgM, IgG, IgA, IgD and IgE, of which IgG is further IgG ₂ , IgG ₃ and I.
It is subclassified into subclasses such as gG ₄ . Among immunoglobulins, IgM has the highest molecular weight and is about 90
It is 5 times larger than IgG, which is 10,000, and is generally considered to correspond to the pentamer of IgG. That is, IgM is a glycoprotein which generally consists of 10 heavy chains, 10 light chains, and 1 J chain, has 10 antibody binding sites, and is further bound with glucosamine oligosaccharide. IgM is considered to be the earliest antibody produced in the immune response. Ig which is a pentamer
Since M stimulates the complement system of animals more efficiently than IgG class antibodies when bound to an antigen, it is known to cause hemagglutination, hemolysis, lysis, neutralization, agglutination with antigen, etc. Has been.

Since this IgM has a high specificity for polysaccharides, it has recently been attempted to be used for cancer diagnosis as an antibody specific to a cancer-related antigen sugar chain. When such IgM is labeled with an enzyme and is applied to an enzyme immunoassay, the labeled IgM antibody becomes a very large polymer and non-specific adsorption at the time of measurement becomes high, resulting in a problem of reproducibility of measurement. It is known that the sensitivity also decreases. On the other hand, as described above, IgM is intended to be used as an immunoassay reagent by utilizing its large antibody titer that it reacts with bacterial antigens and viral antigens even at a very low concentration. In particular, the measurement of a specific IgM antibody generated by an immune reaction in a living body in the acute phase is drawing attention because it is useful for diagnosing an initial infection such as a virus infection or a pathogen infection. A typical example of the immunological measurement method using this IgM measurement is an IgM antibody capture measurement method, and for example, hepatitis A virus (Hepatitis Avi).
rus: HAV) infection diagnosis, Hepatitis B virus core antigen (Hepatitis B virus core)
Antigen: HBc), rubella, measles, mumps, etc.

[0004] In the immunological assay utilizing this IgM assay, an anti-human IgM antibody reagent is usually used to immunologically assay a specific IgM antibody against a target antigen in a test sample. However, at this time, the necessary measurement range of the test sample can be easily set by diluting the test sample with at least IgM or an aqueous solution containing IgM, and specific IgM such as HAV-related antibody can be obtained earlier. A method suitable for an automated measuring system has been found which enables the detection of a. However, when using IgM itself as a reagent, there was a problem that the IgM was unstable. Further, since IgM is a polyvalent antibody, it has been observed that it has a function of reacting with antigens such as bacteria and viruses and erythrocytes to cause aggregation even at a very low concentration. Furthermore, since IgM is a huge molecule compared to IgG and the like, it tends to aggregate, and it is generally considered relatively difficult to stabilize it in a purified form.

In particular, when IgM itself is used as a reagent and, for example, a specimen sample is diluted, IgM is unstable in a dilute solution, and when it is used as a dilute solution, it easily aggregates, which adversely affects the measurement. was there. As described above, IgM is generally very unstable and easily aggregates and precipitates due to various physical or chemical stresses, which is problematic for use as a reagent. Such an unstable IgM must be stored as a concentrated solution or a dry powder and diluted immediately before use. However, this requires an adjusted IgM diluent with a specific concentration for each measurement. There were problems such as difficulty in storing the period.

[0006]

DISCLOSURE OF THE INVENTION The inventor of the present invention has no problem as described above, and is stable as a dilute solution for a long period of time, and is particularly useful as an immunoassay reagent, particularly HAV.
When detecting related antibodies, the required measurement range of the test sample can be easily set and when the specific IgM antibody is detected, the dilution ratio of the pre-dilution is reduced to enable a wider range of measurement. As a result of earnest research to obtain an IgM reagent useful for the measurement system, it was found that these problems can be solved by a simple method, and the present invention was completed.

The present invention relates to an IgM reagent for use in an immunoassay, wherein IgM stabilized by adding bovine serum albumin solution as a stabilizer.
Alternatively, an IgM-containing aqueous solution is obtained, and the stabilized IgM or the IgM-containing aqueous solution is used as the IgM reagent to provide an IgM reagent for immunological measurement. In a more specific aspect, the invention provides anti-human I
In a method for immunologically measuring a specific IgM antibody against a target antigen in a test sample using a gM antibody reagent, at least Ig stabilized with bovine serum albumin solution
The present invention provides a method for measuring a specific IgM antibody, which comprises diluting a test sample with a reagent composed of an aqueous solution containing M or IgM.

The present invention further stabilizes a labeled IgM antibody reagent obtained by labeling with a labeling agent used therein in a method for immunologically measuring an antigen in a test sample with a bovine serum albumin solution. An immunological assay method characterized by the above and a reagent used therefor are provided. In a more specific aspect, a complex comprising the target antigen, the first antibody, and the second antibody by contacting a test sample containing the target antigen with a first antibody and a second antibody In the immunological assay method including the step of forming a compound, one of the first antibody and the second antibody is IgM or an aqueous solution containing IgM, and the IgM is labeled with a labeling agent. In the assay system, the IgM is stabilized with bovine serum albumin solution. For example, in the antigen assay system by the sandwich assay method, the labeled IgM antibody reagent obtained by labeling with a labeling agent can be stabilized by adding bovine serum albumin solution to the assay system, or with bovine serum albumin solution. The label I as a stabilized reagent
A gM antibody reagent can be used in the assay system.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a reagent solution containing IgM,
Although not particularly limited, animal serum, for example, human serum, ascites fluid of an animal into which a hybridoma is transplanted, a culture solution of hybridoma and lymphocyte, a culture solution secreted IgM-like antibody by genetic engineering, or a purified IgM And so on. The origin of IgM is not particularly limited, and examples thereof include animals such as humans, mice, rats, rabbits, goats, sheep, horses, and cows. Use of antisera, polyclonal antibodies, monoclonal antibodies, and mixtures thereof. Can be done.

As a reagent for stabilizing such IgM, bovine serum albumin (BSA) solution can be mentioned.
BSA is about 0.001% to about 25% in the IgM solution.
It can be added in an amount of weight / volume (w / v), preferably about 0.01 to about 20% weight / volume (w / v), more preferably about 0.05 to about 10% weight. / Volume (w / v) amount, more preferably about 0.2 to about 5.0
% Weight / volume (w / v) amount, particularly preferably about 0.5
To about 3.0% weight / volume (w / v), but it can be added in an amount within the range in which stability sufficient for practical use can be ensured and no adverse effect is exerted on measurement. You can choose. BSA may also be added in an amount of about 0.75 to about 2.5% weight / volume (w / v) in the IgM solution. The thus-stabilized IgM can be further labeled if necessary. For example, it can be labeled with a radioisotope such as radioactive iodine, an enzyme such as peroxidase, alkaline phosphatase, galactosidase, etc., acridinium salt, luminescence such as fluorescein or a fluorescent label, and further labeling with biotin or the like. . The thus-stabilized IgM can be further used in a conventional immunoassay. As the immunological measurement method, various methods are known according to the label used, the measurement method, etc.,
For example, a radioimmunoassay, an enzyme immunoassay, a fluorescent immunoassay, a chemiluminescent immunoassay, an agglutination immunoassay, a sandwich method, a competitive method and the like can be mentioned.

In a more specific aspect, the present invention comprises:
The sample is diluted to some extent with an aqueous solution such as a buffer, a diluent or a diluent, and then the sample is diluted with at least bovine serum albumin-stabilized IgM or IgM-containing aqueous solution, and then the antivirus in the sample is diluted. An IgM antibody having specificity for a specific antigen such as a specific IgM antibody is used as an anti-human Ig
I in a sample by reacting with a solid-phase carrier coated with M antibody
The gM antibody is immunologically reacted with the solid phase anti-human IgM antibody, and (a) is then immunologically reacted with a specific antigen reagent such as a virus, and the resulting reaction product is a chemiluminescence-labeled antiviral antibody. Of a specific IgM antibody, which comprises reacting a labeled antibody such as H., etc. immunologically, or (b) reacting a labeled antigen labeled with a specific antigen reagent such as a virus, immunologically Methods and reagents used in the measurement methods are provided. In yet another specific aspect, the present invention provides the test sample containing a target antigen by contacting the first antibody and the second antibody with the target antigen, the first antibody, and the second antibody. In the immunoassay method including the step of forming a complex consisting of the antibody of 1), an IgM reagent labeled with a labeling agent is used as one of the first antibody and the second antibody, In the method, the labeled IgM reagent is stabilized with at least bovine serum albumin solution.

More preferably, in the method, the test sample comprises a first antibody (immobilized antibody) bound to a solid phase carrier for an antigen of interest in the test sample and a second labeled antibody.
Contact with an antibody (labeled IgM antibody),
Measurement of forming a complex of an antibody, the antigen, and the labeled IgM second antibody, separating the complex from the unreacted labeled antibody, and then measuring either the labeled antibody or the unreacted labeled antibody in the complex In the system, the labeled IgM antibody reagent is stabilized by allowing a bovine serum albumin solution to coexist in the assay system. The contact between the target antigen in the test sample and each antibody may be such that the immobilized first antibody and the labeled IgM second antibody are simultaneously contacted with the target antigen in the test sample. Alternatively, first, the target antigen in the test sample may be brought into contact with the solid-phased first antibody, and if necessary, after washing treatment, the labeled IgM second antibody may be brought into contact. Alternatively, the target antigen in the test sample may be first contacted with the labeled IgM second antibody, and then the solid phase immobilized first antibody may be contacted. Typically, various methods widely known as the sandwich method can be applied. For example, in the antigen assay system by the sandwich assay method,
Preferably, the labeled IgM antibody can be stabilized by adding bovine serum albumin solution to the assay system, or the labeled IgM antibody stabilized with bovine serum albumin solution.
It can be used as an antibody reagent in the measurement system.

An example of a particularly preferred measurement system is HAV.
A method for diagnosing hepatitis A infection by measuring an IgM anti-HAV antibody that occurs in the acute phase of infection can be mentioned. 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. 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 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 elderly people. From the situation that many positive people are distributed,
Accurate and quick HAV in the sense of preventing the HAV infection
It is required to detect the presence or absence of infection. Since HAV uses oral infection by feces as its main transmission route, there is a great risk of infection in areas with poor environmental hygiene, and the number of overseas travel opportunities has increased recently. It is also important in terms of preventing infections between employees and employees.

In order to detect HAV infection in this acute phase, an anti-HAV antibody, particularly 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 a means for solving this, for example, an aqueous solution of human IgM-containing fraction, purified human IgM, or the like is added to a sample, and an IgM antibody specific to an antigen of interest can be measured without being affected by the amount of IgM in the sample. To do so.

When the test sample is diluted with the IgM-containing solution, the test sample may be appropriately diluted with physiological saline or the like in advance. Furthermore, by adding the human IgM solution, it is possible to achieve a wider range of measurement, and it becomes possible to easily set the measurement range, such as the preparation of the measurement sample, for example, the adjustment of the sample concentration. The application in the measurement system becomes easy. However, since the IgM solution is unstable, a more stable IgM solution is preferable as a reagent. In the present invention, human Ig stabilized with bovine serum albumin solution
It has been recognized that similar advantages can be obtained by adding an M-containing fraction and an aqueous solution of purified human IgM stabilized with a bovine serum albumin solution. Thus, as described above, specific IgM, for example, a HAV-related antibody can be detected, or the dilution ratio of the pre-dilution can be reduced to allow a wider range of measurement, and for example, reliable measurement in a high concentration region such as type A liver disease. Various measurement methods become possible.

The HAV antigen reagent used in the system for specifically measuring the IgM anti-HAV antibody according to the present invention uses a viral antigen obtained by an in vitro cell culture method. 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-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'
In a growth medium such as balanced salt solution), if necessary, antibiotics such as fetal calf serum (FCS), penicillin, streptomycin, yeast extract, bactopeptone, lactalbumin hydrolyzate,
Cultivated in the addition of other cell growth factors,
It is then obtained from the cell culture thus obtained as follows.

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. As described above, the HAV extract 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 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) 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. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (β-aminoethyl ether) -N, N,
N ', N'-tetraacetic acid (EGTA) and the like.

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.

As the anionic surfactant, alkali metal salt or alkaline earth metal salt of higher fatty acid having 12 to 18 carbon atoms such as potassium stearate, alkali metal salt of bile acid, higher fatty acid having 12 to 18 carbon atoms. Organic salts such as triethanolamine, lithium dodecyl 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 sulfonate, 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. These surfactants
The amount used is preferably selected according to the amount of coexisting protein, for example, about 0.001% v / v to about 10% v /
It can be used in the range of v. Particularly preferably SDS
Of about 0.05% v / v to about 5% v / v, more preferably about 0. 0% when no other proteins coexist.
It can be used in the range of 5% v / v to about 1.0% v / v.

In treating the HAV extract with a surfactant, the HAV extract may be 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 before 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 about 0.5% v / v of HAV extract obtained from cell lysate obtained by in vitro cell culture method. 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 antigen reagent can be obtained from a virus culture, or can be a recombinant antigen expressed in Escherichia coli, yeast or the like using a gene recombination technique.

In measuring the specific IgM antibody in the sample in the present invention, the anti-IgM antibody may be, for example, agar, agarose, crosslinked agarose, crosslinked alginic acid, cellulose, nitrocellulose or carboxycellulose, if necessary. Cellulose ester or mixed cellulose ester, paper, dextran, gelatin, cross-linked gelatin, chitin, collagen, cotton or other bio-derived or naturally-derived polymer, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate , Polyethylene, polypropylene, polyvinyl alcohol, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polymethacrylate,
Acrylic resins such as polyacrylamide, ion exchange resins, photocrosslinking resins, polyesters, polyamides, synthetic polymers and resins such as polyacetals, natural or synthetic modified or unmodified polymerized carbohydrates, polymerized hydrocarbons, and other crosslinked derivatives thereof. , Glass, such as activated glass, silica gel, alumina, silica-alumina,
Fine particles made of inorganic materials such as barium sulfate, ceramics, carbon and magnesium sulfate, beads, microplates, microtiter wells, microtubes, strips, membranes, trays, gels, etc., as well as red blood cells, rubber, latex particles, emulsions, etc. Of the specific IgM antibody as an analysis target, and the solid phase of the anti-IgM antibody thus immobilized and the specific IgM antibody in the analysis sample. Specific binding reaction of the anti-Ig
This can be done by detecting the specific IgM antibody bound to the M antibody.

In a preferred embodiment, the anti-human IgM antibody-bound solid phase reacted with a sample in the present invention is
Beads made of polystyrene, fine particles made of polystyrene, and the like can be used. Moreover, as an antibody, human I
Any antibody to gM can be used without particular limitation. Antibodies can be obtained by a conventional method, for example, Shigeru Muramatsu, et al., Laboratory of Experimental Biology 14, Immunobiology,
Maruzen Co., Ltd., 1985, edited by The Biochemical Society of Japan, Sequential Biochemistry Experiment Course 5, Immunobiochemistry Research Method, Tokyo Kagaku Dojin, 198
6 years, Japan Society for Biochemistry, Shinsei Chemistry Laboratory 12, Molecular Immunology III, Antigen / Antibody / Complement, Tokyo Kagaku Dojin, 199
According to the method described in 2 years, for example, it can be obtained by immunizing horse, cow, sheep, rabbit, goat, rat, mouse or the like, or can be a monoclonal antibody.
These may be used alone or in combination with each other. These antibodies, if necessary, pepsin,
Digested with enzymes such as papain to give F (ab ') ₂ , Fa
It may be used as b. As an anti-human IgM antibody,
Preferred examples include an antibody that specifically reacts with the μ chain and an anti-μ chain antibody, which may be a monoclonal antibody obtained by utilizing a cell fusion technique using mouse myeloma cells. There is no end. In the case of an antibody against a target antigen, it can be prepared, immobilized, modified, purified, or a monoclonal antibody can be prepared in the same manner as 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 an antibody labeled with a colored substance such as colloid or colored latex particles is used as a reagent, and the antibody or the antigen in the analytical sample is directly or indirectly bound to cause a radioactivity,
By measuring enzyme activity, chemiluminescence or the presence or absence of color,
It is possible to determine whether or not the antibody or the like was present in the sample. In the present invention, in particular, chemiluminescence labeling method, for example, acridinium ester or fluorescence labeling method, for example, chemiluminescence or fluorescence immunoassay using an antibody reagent labeled with fluorescein or the like can perform automated measurement. This is the preferred method. Particularly, the chemiluminescence immunoassay method using an antibody reagent labeled with an 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.

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 an acridinium label, treatment with a coloring reagent, for example hydrogen peroxide, such as about 0.01% to
The measurement can be performed by using a luminometer or the like after treating with about 0.1% hydrogen peroxide aqueous solution and sodium hydroxide, for example, about 0.05 N to about 0.5 N sodium hydroxide aqueous solution.

Of course, the labeling agent is not limited to the above-mentioned ones, and is appropriately selected from those known to be used in the relevant field in consideration of the equipment used for the measurement, the place, etc. according to the purpose. Can be used.

For binding or adsorbing the solid phase or the label with the antigen or the antibody, a method generally used in the art can be used. For example, physics such as ionic interaction, hydrophobic interaction, covalent bond and the like can be used. It can be performed by dynamic adsorption or chemical bonding. For example, as a cross-linking agent, glutaraldehyde, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, N, N'-o-
Phenylene dimaleimide, 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.

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 used. As a surfactant, polyoxyethylene sorbitan (typically, Tween 2
0, etc.), polyoxyethylene ether (a typical one is available under a trade name such as Triton X-100), an octylphenol-ethylene oxide condensate (a typical one is Nonide
(available under trade names such as tP-40) and the like. As the buffer, diluent or diluent, water, phosphate buffer, Tris buffer, physiological saline, etc. as described above, HEPES solution, PBES solution, CAPS solution, MOPS
Liquid, amino acid liquid and the like. These alone,
It can also be used by optionally blending. 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 a human IgM solution stabilized with an appropriate amount of bovine serum albumin solution and an anti-human IgM antibody-bound solid phase carrier or particulate carrier are reacted, and then (1) HAV infection HAV extract harvested from cultured cells or (2) this HAV extract with at least a surfactant,
For example, it is immunologically reacted with a HAV antigen obtained by treating with SDS, and the obtained reaction product is chemiluminescence-labeled anti-HA.
A method for measuring a HAV antibody, which comprises immunologically reacting a V antibody, for example, an acridinium-labeled anti-HAV antibody, and then performing detection after reacting with a trigger reagent consisting of a hydrogen peroxide solution and a sodium hydroxide solution. Can be done.

In the present invention, it can be used in a known immunoassay 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, chickenpox virus, mumps,
Examples include measles, rubella, AIDS virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and the pathogenic microorganisms include Shigella dysenteriae. ), B. pertussis (Bordetella perussis) and the like.

[0039]

EXAMPLES Next, the present invention will be described more specifically with reference to examples. However, it is 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. Should be. Example 1 Preparation of IgM Stabilized with Bovine Serum Albumin Solution Commercially available human IgM solution (manufactured by Chemicon International, Inc. [Chemicon International]
al, USA. ]) Was diluted in 0.01 M Tris-HCl buffer (pH 8.5) containing 1% bovine serum albumin and 0.9% sodium chloride and 0.1% sodium azide. (75 μg / ml I
gM). Stabilized with the obtained bovine serum albumin solution I
The gM solution was stored at 4 ° C. for various times and used as a human IgM reagent for dilution.

Preparation of Anti-Human μ-IgM Antibody-Coated Microparticles A polyclonal antibody (Jackson ImmunoRese manufactured by Jackson ImmunoResearch Laboratories, Inc., USA, which 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 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 IgM type HAV antibody positive panel samples were diluted with saline. The diluted sample was further diluted 5-fold with IgM reagent (30 μl) stabilized with bovine serum albumin solution stored at 4 ° C. for various times. As a comparison, after the IgM type HAV antibody negative panel sample was diluted with physiological saline, the bovine serum albumin solution-free IgM-containing buffer (each 0.0
1M Tris buffered saline (pH 8.5), 0.0
1 M Tris buffer (pH 8.5), 0.1 M Tris buffer (pH 8.5), 0.01 M Tris buffer (p
It was further diluted 5-fold with H7.2)). The diluted sample (125 μl) was placed in a container, to which anti-human μ-I was added.
Add the gM antibody-coated microparticle reagent (30 μl) and incubate at 37 ° C.
For 20 minutes. The reacted fine particles were captured by a glass fiber filter, and a 0.1 M borate buffer solution (pH 8.
5) (300 μl) was washed twice. Next, add HAV antigen reagent (30 μl) to the surface of the filter,
It was reacted with the fine particles captured on the filter surface for 0 minutes. The filter was washed with 0.1 M borate buffer (pH 8.
5) (100 μl) once and the same buffer (300 μl)
Washed once in l). Next, acridinium labeled anti-HAV
Add antibody reagent (30 μl) to the filter surface and
It was made to react with the fine particles captured on the filter surface at 10 ° C. for 10 minutes. Filter with 0.1 M borate buffer (p
H8.5) (100 μl) once and the same buffer (3
Washed once with 00 μl).

The filter was transferred to a chemiluminescence reader in which a coloring solution containing 0.4% hydrogen peroxide in 0.25 N NaOH (50 μl) was pumped into the filter. Acridinium bound to the microparticles emitted light and the amount of light generated was measured. The results are shown in FIG. With the IgM reagent containing no bovine serum albumin, I
An increase in luminescence (photon count) due to aggregation of gM molecules is observed, but Ig stabilized with bovine serum albumin solution
With the M reagent, the amount of luminescence (photon count) does not substantially increase with the lapse of storage time.

Example 2 Preparation of SDS-treated HAV Antigen HAV was prepared 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. 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 HAV-M positive panel samples were diluted with saline in the same manner as in Example 1. The diluted sample was further treated with IgM reagent (30 μl) stabilized with bovine serum albumin solution for 5 times.
Diluted 1-fold. As a comparison, after the HAV-M negative panel sample was diluted with physiological saline, it was further diluted 5-fold with bovine serum albumin solution-free IgM reagent. The diluted sample (125 μl) was placed in a container, to which anti-human μ-Ig was added.
M antibody-coated microparticle reagent (30 μl) was added, and the mixture was reacted at 37 ° C. for 20 minutes. The reacted fine particles were captured by a glass fiber filter, and a 0.1 M borate buffer solution (pH 8.
5) (300 μl) was washed twice. Next, SDS processing H
Add AV antigen reagent (30 μl) to the filter surface,
It was allowed to react with the fine particles trapped 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 chemiluminescence reader in which 0.25 N Na
Luminescent solution containing 0.4% hydrogen peroxide in OH (50μ
l) was sent to the filter. Acridinium bound to the microparticles emitted light and the amount of light generated was measured. The same results as in Example 1 were obtained.

Example 3 In the same manner as in Example 1, the HAV-M positive panel sample was diluted with physiological saline. The diluted samples were further diluted to various concentrations with IgM reagent stabilized with bovine serum albumin solution. As a comparison, after the HAV-M negative panel sample was diluted with physiological saline, bovine serum albumin solution-free IgM was added.
Diluted with reagents. The diluted sample (125 μl) was placed in a container, anti-human μ-IgM antibody-coated microparticle reagent (30 μl) was added thereto, and the mixture was reacted at 37 ° C. for 20 minutes. Capture the reacted fine particles with a glass fiber filter,
0.1 M borate buffer (pH 8.5) (300 μl)
And washed twice. 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 the filter with 0.1 M borate buffer (pH 8.5) (1
00 μl) and once with the same buffer (300 μl)
Washed twice. Next, an acridinium-labeled anti-HAV antibody reagent (30 μl) was added to the filter surface, and the mixture was added at 37 ° C. for 10 hours.
It was allowed to react with the fine particles captured on the filter surface for a minute. The filter was washed with 0.1 M borate buffer (pH 8.
5) (100 μl) once and the same buffer (300 μl)
Washed once in l).

The filter was transferred to a chemiluminescence reader, in which a luminescent solution (0.4 μl) containing 0.4% hydrogen peroxide in 0.25 N NaOH was pumped into the filter. Acridinium bound to the microparticles emitted light and the amount of light generated was measured. By diluting with a human IgM-containing solution stabilized with bovine serum albumin solution, it was found that the effect can be obtained by using a smaller amount of the IgM-containing solution.
It can be seen that in such a measurement system, the IgM reagent stabilized with the bovine serum albumin solution can be stored and convenient. It is unexpected to show such excellent properties as a reagent in immunological measurement.

[0049]

INDUSTRIAL APPLICABILITY In the measurement of IgM antibody in a sample, the test sample is stabilized with at least bovine serum albumin solution in order to avoid the limitation of the amount of diluent, obtain a necessary measurement range and perform better measurement. By diluting with the prepared IgM or the IgM-containing aqueous solution, it became possible to assemble a stable and wide range of measurement system which is advantageous for automation. IgM or I stabilized with bovine serum albumin solution
The gM-containing aqueous solution has excellent storage stability, can be easily used, and has a greater effect than IgM to which bovine serum albumin solution is not added. The IgM-containing aqueous solution reagent does not need to be diluted or prepared for each measurement, and the once prepared IgM-containing aqueous solution can be used for the measurement again.
Since bovine serum albumin can be obtained in a large amount and stably, an inexpensive IgM reagent for immunological measurement can be obtained.

[Brief description of the drawings]

FIG. 1 Luminescence in immunoassay when diluted with IgM diluent stabilized with bovine serum albumin solution and after diluted with IgM diluent without bovine serum albumin solution after storage for various times. The relationship in quantity is shown.

Claims (13)

[Claims] 1. An IgM reagent for immunological measurement, which is an IgM reagent or an aqueous solution containing IgM stabilized with bovine serum albumin solution, for use in an immunological measurement method. 2. The amount of bovine serum albumin present in the stabilized IgM or IgM-containing aqueous solution is about 0.05-.
The IgM reagent for immunoassay according to claim 1, which is in an amount of about 10% weight / volume (w / v). 3. The amount of bovine serum albumin present in the stabilized IgM or IgM-containing aqueous solution is about 0.75.
The IgM reagent for immunoassay according to claim 1, which has an amount of about 2.5% weight / volume (w / v). 4. The IgM reagent for immunological measurement according to any one of claims 1 to 3, wherein the IgM reagent is labeled IgM labeled with a labeling agent or a labeled IgM-containing aqueous solution. 5. The immunological assay according to claim 4, wherein the label is selected from the group consisting of radioisotopes, enzymes, luminescent substances, fluorescent substances, and biotin. IgM reagent. 6. The IgM reagent for immunological measurement according to claim 4 or 5, wherein the labeled IgM reagent is used in an antigen measurement system by a sandwich assay method. 7. A method for immunologically measuring a specific IgM antibody against a target antigen in a test sample using an anti-human IgM antibody reagent, comprising at least human IgM stabilized with bovine serum albumin or human. A method for measuring a specific IgM antibody, which comprises diluting a test sample with a reagent composed of an IgM-containing aqueous solution. 8. A specific IgM antibody is an Ig against HAV.
The method for measuring a specific IgM antibody according to claim 7, which is an IgM antibody against M antibody or HBc. 9. (1) IgM or I, which is obtained by diluting a sample to be measured with a buffer, a diluent or an aqueous solution of a diluent as necessary, and then stabilized with at least bovine serum albumin solution.
After diluting the sample with a reagent consisting of an aqueous solution containing gM,
The target-specific IgM antibody in the sample is reacted with an anti-human IgM antibody-bound solid phase carrier to cause the IgM antibody in the sample to immunologically react with the solid phase anti-human IgM antibody, (2) (a) The reaction product thus obtained is immunologically reacted with a specific antigen reagent, and a labeled antibody obtained by labeling an antibody against the antigen reagent with a labeling agent is immunologically reacted, or (b) the obtained reaction product 9. The method for measuring a specific IgM antibody according to claim 7, wherein a labeled antigen obtained by labeling the specific antigen reagent with a labeling agent is reacted immunologically. 10. A complex comprising the target antigen, the first antibody, and the second antibody is formed by contacting a test sample containing the target antigen with a first antibody and a second antibody. In the immunological measurement method including the step of using an IgM reagent labeled with a labeling agent as either one of the first antibody and the second antibody, the method comprises using at least bovine serum albumin solution in the method. A method comprising stabilizing a labeled IgM reagent. 11. The test sample comprises a first antibody (immobilized antibody) bound to a solid phase carrier for a target antigen in the test sample and a labeled second antibody (labeled antibody). 11. A complex of the first antibody, the antigen, and the second antibody is formed by contacting with, and either the labeled antibody or the unreacted labeled antibody in the complex is measured. the method of. 12. (i) (a) Binding a target antibody to the first immobilized antibody by contacting a test sample containing the target antigen with a first antibody bound to a solid phase carrier After washing the solid phase as necessary, an immune complex is formed by contacting with at least a second antibody which is labeled IgM stabilized with a bovine serum albumin solution, or (b) the target antigen is The target antigen is bound to the second labeled antibody by contacting the test sample containing it with a labeled and stabilized IgM second antibody, and then the first antigen bound to a solid phase carrier. An immune complex is formed by contacting the antibody, and (ii) the solid phase is washed if necessary, and either the labeled antibody or the unreacted labeled antibody in the complex is measured. Item 10 or 12. The method according to 11. 13. The method according to claim 7, wherein the sample to be measured is whole blood, serum, or plasma.