JP2003043044A - Immunoassay method for antibody by antibody capturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunoassay method for antibody by an antibody capturing method wherein an antigen to which a tag is bonded and the antibody with reference to a labeled tag are used and to provide an antibody measuring reagent. SOLUTION: In the immunoassay method for antibody with reference to the antigen in a specimen by the antibody capturing method, at least the antigen to which the tag is bonded and the labeled antibody with reference to the tag are used. In the antibody measuring reagent in the specimen, at least the antigen to which the tag is bonded and the antibody with reference to the labeled tag are contained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immunologically measuring an antibody by an antibody capturing method and an antibody measuring reagent, which uses an antigen bound to a tag and an antibody against the labeled tag. The immunological measurement method of an antibody by the antibody capture method and the antibody measurement reagent can be used for diagnosing an infectious disease.

[0002]

2. Description of the Related Art An immunological measurement method is one of the useful methods for diagnosing infectious diseases and the like. As an diagnostic method for infectious diseases, measuring an antibody against a pathogen present in a sample such as serum is one of important means. There are 5 subclasses of antibody, and IgG, IgM, IgA for the diagnosis of infectious diseases.
Is often measured. When measuring IgG in a sample using an immunological measurement method, the antigen is solid-phased on a plate, the sample is added thereto, and after adding IgG to the antigen,
It is general to use a so-called antigen solid phase method, which recognizes human IgG and detects bound IgG using a secondary antibody labeled with an enzyme. On the other hand, if the solid phase antigen method is used to measure IgM, which has a lower content compared to IgG, IgG and IgM compete for binding to the solid-phased antigen, resulting in decreased sensitivity. A problem arises. Regarding infectious diseases, the amount of IgM and IgG in blood is different between acute and chronic infectious diseases, so it is necessary to measure IgM and IgG by class. There was a problem that it could not be measured in. Further, in the antigen solid phase method, a rheumatoid factor or the like may be erroneously detected, which may lead to a decrease in specificity.

Further, instead of directly immobilizing the antigen on the plate, an antibody against the antigen is immobilized on the plate, the antigen is added, and then a sample is added, which further recognizes human IgM and is labeled with an enzyme. IgM bound using a secondary antibody
The method of detecting is also commonly used. However, even in this method, the problem of reduced sensitivity due to competition over the binding of IgG and IgM to the antigen when measuring IgM, the problem of false detection due to the rheumatoid factor cannot be avoided, and furthermore, it differs for each antigen. The antibody must be immobilized on the plate,
There was also the problem of high costs.

[0004] In order to solve these problems, an operation of treating the sample with an IgG absorbent may be added in advance, but it is difficult to obtain a complete effect. Therefore, ELISA (enzyme-linked immunosorbent assa)
y) is being implemented.

Antibody-measuring method When measuring IgM antibody by ELISA, first, a plate on which anti-human IgM antibody is solid-phased is prepared and a sample is added. IgM in the specimen is solid human anti-human IgM
It binds to the antibody and is captured. Next, when an antigen is added, IgM that specifically recognizes the antigen among the captured IgM binds to the antigen. Finally, the captured IgM is detected using a secondary antibody that binds to this antigen and is labeled with an enzyme. By this method, competitive reaction with IgG and nonspecific reaction due to rheumatoid factor and the like can be avoided. Although other types of labeling substances such as radioisotopes (RI) are used instead of enzymes, the basic principle is the same.

As a result of recent advances in gene recombination techniques, the number of cases in which antigens used in immunological measurement methods are produced by gene recombination methods has increased. The method of culturing a pathogen and purifying the antigen from the pathogen has the problems that the worker is at risk of infection and the cost is high. These problems can be solved by extracting a gene encoding an antigen from a pathogen, incorporating it into an expression vector gene, introducing it into Escherichia coli or various animal cells, and expressing it in the body. Furthermore, expressing only a part of the antigen, or expressing it as a fusion protein with other antigens to increase reactivity,
GST (Glutathiol) for use in affinity purification, etc.
neS-Transferase) and various other methods such as expression as a fusion protein with a protein.

Utilization of these fusion proteins for immunological assay has also been attempted. JP-A-8-62217 discloses that instead of immobilizing an antibody against the aforementioned antigen on a plate, a fusion protein of the antigen and a carrier protein such as GST is prepared in advance, and an antibody against the carrier protein is prepared on the plate. An immunological assay, which is solid phased, is described. The fusion protein is captured on the plate by the binding of the antibody against the carrier protein and the carrier protein, and the antibody against the antigen can be measured by adding the sample and the labeled secondary antibody thereto. By this method,
It becomes possible to carry out immunological measurement without producing different antibodies for each antigen. However, since this method is not an antibody capture method, it is necessary to solve the problem of sensitivity decrease due to competition over binding of IgG and IgM to the antigen when measuring IgM, and the problem of false detection due to rheumatoid factor. I haven't arrived.

[0008]

[Problems to be Solved by the Invention] Antibody capture method for detecting antibodies in a sample ELISA and a method similar thereto
Although it became possible to avoid competitive reactions with gG and non-specific reactions due to rheumatoid factors, etc., these methods still have some problems to be improved.

First, there is a problem in producing an antibody for labeling. The antibody in the sample and the labeled antibody bind to the antigen, but the labeled antibody must be such that the binding site in the antibody with the antigen in the sample does not compete as much as possible. Otherwise, the sensitivity will drop significantly. However, when the epitope part in the antigen is limited, it may be difficult to select such an antibody.

The second is the problem of inactivation during antibody labeling.
When labeling an enzyme or fluorescent substance, the two are bound through the reactive group of the amino acid side chain in the antibody, but this causes a change in the ability of the antibody to bind to the antigen, and in some cases the binding power is significantly reduced. In some cases.

Thirdly, the antibody capture method ELISA is an antibody solid phase method ELI.
The number of steps is one more than that of SA, and the required time is longer. In particular, this is a problem when it is necessary to know the presence or absence of infection as soon as possible, such as in emergency medical care. In addition, this results in an increase in cost for an inspection center that conducts medical tests at the request of medical institutions. Therefore, it has been desired to develop a method capable of solving the above problems.

[0012]

Means for Solving the Problems As a result of intensive studies by the present inventors, a tag or a carrier (hereinafter referred to as a tag)
It is possible to overcome the above-mentioned first to third problems by carrying out an antibody immunological assay method in a sample by an antibody capture method using an antigen bound with a substance such as a protein and a labeled antibody against the tag. Heading, completed the present invention. In particular, it is preferable to mix the antigen and the labeled antibody before adding them into the wells of the immunoassay plate.

According to the present invention, a sample is added to an immunological assay plate for capturing an antibody in the sample, for example, an anti-IgM antibody solid phase plate, an antigen having a tag bound thereto is added, and then the tag is labeled. Mixing and adding antibodies,
Preferably, the step of adding the sample to an immunological assay plate that captures the antibody in the sample, for example, an anti-IgM antibody solid phase plate, and then mixing and adding the tag-bound antigen and the labeled antibody to the tag Characterized by having,
It is intended to provide a method for immunologically measuring an antibody by an antibody capture method and a reagent for measuring IgM in a sample using this method.

That is, the present invention is as follows. (1) A method for immunologically measuring an antibody against an antigen in a sample by an antibody capture method, which comprises using an antigen bound to at least a tag and a labeled antibody against the tag. (2) An antibody against the antigen in the sample by the antibody capturing method according to (1), which further comprises a plate on which a substance that binds to the antibody in the sample against the antigen bound with the tag is immobilized. Immunological measurement method. (3) (a) adding a sample to a plate on which a substance that binds to an antibody is immobilized, (b) adding an antigen having a tag bound to the plate, and (c) adding the tag to the plate A method for immunologically measuring an antibody against the antigen in a sample by the antibody capturing method according to (1) or (2), which comprises the step of adding a labeled antibody against the antigen. (4) The immunological measurement method is ELISA, (1) to
The immunological measurement method according to any one of (3). (5) The method for immunochemically measuring an antibody by the antibody capture method according to any one of (1) to (4), wherein the antigen bound with the tag and the antibody against the labeled tag are mixed in advance and then used.

(6) The antibody according to any one of (1) to (5), wherein the antigen bound with the tag is a fusion protein of the tag and the antigen, which is produced by a gene recombination method. Immunological measurement method of antibody by capture method. (7) Tag is His ₆ , GST (Glutathione S-Transferas
e), MBP (Maltose Binding Protein), a galactose binding protein, and a protein selected from the group consisting of thioredoxin, characterized in that the immunology of the antibody by the antibody capture method according to any one of (1) to (6) Measurement method. (8) The antigen is Epstein-Barr virus (EBV)
An immunological assay method for an antibody by the antibody capture method according to any one of (1) to (7), which is an antigen. (9) Immunization of an antibody against the antigen in a sample by the antibody capture method according to any one of (1) to (8), wherein the substance that binds to the antibody in the sample is an anti-human immunoglobulin antibody Measurement method. (10) The method for immunologically measuring an antibody by the antibody capture method in a sample according to (9), wherein the substance that binds to the antibody in the sample is an anti-human IgM antibody.

(11) A reagent for measuring an antibody in a sample by an antibody capturing method, which comprises at least an antigen bound to a tag and an antibody against the labeled tag. (12) Further, it includes a plate on which a substance that binds to an antibody in a sample for the antigen bound to the tag is immobilized.
The antibody measurement reagent according to (11). (13) The antibody measuring reagent according to (11) or (12), wherein the antigen bound with the tag is a fusion protein of the tag and the antigen, which is produced by a gene recombination method. (14) Tag is His ₆ , GST (Glutathione S-Transfer
ase), MBP (Maltose Binding Protein), a galactose binding protein, and a protein selected from the group consisting of thioredoxin, (11) to (13)
5. The antibody measuring reagent according to any one of 1. (15) The substance that binds to the antibody in the sample is an anti-human immunoglobulin antibody, (11) to (1)
The antibody measurement reagent according to any one of 4).

(16) The antibody measuring reagent according to (15), wherein the substance that binds to the antibody in the sample is an anti-human IgM antibody. (17) The antigen is Epstein-Barr virus (EB
V) is an antigen, (11) to (16)
5. The antibody measuring reagent according to any one of 1. Hereinafter, the present invention will be described in detail.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Immunological measurement of antibodies by the antibody capture method of the present invention uses a tag-bound antigen, a labeled antibody to the tag, and a carrier to which a substance that captures the antibody to be measured is bound. The antigen bound with the tag, the labeled antibody against the tag, and the carrier bound with the substance that captures the antibody to be measured can be prepared as follows. Here, the “antibody capture method” refers to a method of capturing an antibody to be measured using a carrier to which a substance that binds to the antibody to be measured is bound, and measuring the antibody.

1. Preparation of antigen bound with tag In the present invention, a "tag" is a substance bound to an antigen to which an antibody in a sample to be measured specifically binds, and a labeled secondary antibody specifically binds to the antigen. Refers to a substance that does.

The tag can be used as long as it has a property of being antigenic and can bind to the labeled secondary antibody by an antigen-antibody reaction. In the present invention, His ₆ (histidine hexamer), GST (Glutathione S-Transferase), MBP (Malto
se binding protein), galactose binding protein, thioredoxin, and other proteins and peptides capable of producing a fusion protein with an antigen protein by gene recombination can be preferably used.

The tag-bound antigen can be prepared by chemically or enzymatically binding a substance serving as a tag and an antigen specifically bound by an antibody to be measured in a sample. When the tag is a protein, a method of producing a fusion protein by expressing a fusion gene of a gene encoding the tag protein and a gene encoding an antigen can be used.

The fusion of the gene encoding the tag protein and the gene encoding the antigen can be carried out by an ordinary gene recombination technique. At this time, it can be performed by introducing an appropriate restriction enzyme recognition and cleavage sequence. The gene encoding the tag protein may be added to the C-terminal side or the N-terminal side of the antigen-encoding gene. At this time, the stop codon should not appear between the gene encoding the tag protein and the gene encoding the antigen. The distance between the gene encoding the tag protein and the gene encoding the antigen is not limited, and a linker may be included between the two. In order for the tag protein and the antigen to be translated, the open reading frames of both genes should be aligned. Further, between both genes, it may have a base sequence such that a specific amino acid is contained in the linker site.

2. Preparation of an antigen to which the antibody to be measured specifically binds The immunological measurement of the antibody of the present invention is intended to measure an antibody against a specific antigen, and the antigen used in the present invention is to be measured. Is an antigen to which the antibody specifically binds. The antigen is not limited to a particular one, and includes, for example, an antigen recognized by an antibody against self that appears in an autoimmune disease, an antigen recognized by a cancer-specific antibody, and an antigen of a pathogen in an infectious disease. In particular, pathogens or constituents of pathogens for the diagnosis of infectious diseases are used as antigens. For example, when the pathogen is bacteria, bacterial proteins such as bacterial proteins, sugar chain antigens on the surface of the bacteria, and lipid antigens derived from bacterial membranes are used.When the pathogen is a virus, the structural proteins and non-structural proteins that make up the virus particle are used. Proteins and the like are used as antigens, the former includes capsid proteins and viral proteins in the envelope, and the latter includes proteins such as nuclear antigens that have functions required for virus replication and proliferation. Examples of pathogens include Epstein-Barr virus (EBV), parvovirus, mumps virus, viruses such as influenza virus, chlamydia, and bacteria such as syphilis. For example, in the case of EBV, EB virus capsid antigen (VCA), EB
Virus-specific nuclear antigen (EBNA), EB virus-associated early antigen (EA), EB virus-associated membrane antigen (MA), EB virus latent infection membrane protein (LMP), etc. are used. Above all, E
The VCA protein of BV can be preferably used in the present invention.

EBV is a causative pathogen of infectious mononucleosis, nasopharyngeal cancer, Burkitt lymphoma and the like, and is a virus which has been recently pointed out to be associated with the development of gastric cancer. It has become more and more important in recent years to grasp the presence or absence of the infection.

The antigen of the present invention can be obtained by culturing the above-mentioned pathogen, or can be obtained by genetic engineering from a specific gene in the genome of the pathogen. At this time, the antigen used may be the pathogen itself or a specific antigen that is a constituent of the pathogen.

3. Preparation of antibody against tag The antibody against the tag used in the present invention can be obtained by immunizing an animal with the tag as an antigen, collecting antiserum, and purifying the antiserum. Alternatively, it can be obtained as a monoclonal antibody using the cell fusion method. be able to. In addition, a commercially available product can also be used (for example, the anti-GST antibody is sold by Amersham Pharmacia Biotech).

4. Labeling of antibody to tag (preparation of labeled antibody) As a labeling substance of antibody to the tag, a fluorescent dye such as fluorescein isothiocyanate (FITC), an enzyme such as peroxidase and alkaline phosphatase, a luminescent substance, a radioactive isotope and the like are used. The method of binding the labeling substance to the antibody to the tag can be carried out by a method known to those skilled in the art. For example, in the case of enzyme labeling, the maleimide method or the like (Eiji Ishikawa “Ultrasensitive enzyme immunoassay”)
The method (described in the academic society publication center etc.) can be used.

5. Preparation of antibody-capturing substance solid-phase carrier The substance that binds to the antibody to be measured can be bound to an appropriate carrier to prepare the antibody-capturing substance solid-phase carrier of the present invention. Examples of the antibody-capturing substance include antibodies against human antibodies, protein A, protein G and the like, and preferably antibodies against human antibodies are used. Antibodies against human antibodies include anti-human IgG antibodies, anti-human IgM antibodies, and anti-human IgA antibodies.

Anti-human IgG antibody, anti-human IgM antibody, and / or
Alternatively, the anti-human IgA antibody can be immobilized on a carrier such as an ELISA titer plate or beads to obtain the antibody solid phase carrier used in the present invention. As the anti-human IgG antibody, anti-human IgM antibody, and anti-human IgA antibody, commercially available products can be used, or human IgG (γ), IgM (μ), and IgA (α) can be immunized in animals to prepare them. You can also The antibody is immobilized on a carrier such as a titer plate by physical adsorption or chemical bonding. The amount of solid phase immobilization is not particularly limited, but when the carrier is a microtiter plate, it is preferably several ng to several tens μg per well. The immobilization can be performed by dissolving the antibody to be immobilized in an appropriate buffer and contacting with a carrier. For example, in the case of using a microtiter well, the antibody solution can be solid-phased by dispensing it into a well of a microtiter plate and leaving it for a certain period of time. Anti-human IgG antibody, anti-human I
The gM antibody and the anti-human IgA antibody can be selected according to the class of the antibody to be measured. In addition, when measuring multiple classes of antibodies at the same time, anti-human IgG antibody,
The anti-human IgM antibody and the anti-human IgA antibody may be solid-phased or a mixture of two or all of them.

6. Preparation of Antibody Measuring Reagent By combining at least the above-described antigen bound with a tag and an antibody against the tag, an antibody measuring reagent for use in detecting an infectious disease, that is, an antibody measuring kit can be prepared. The reagent may further include an antibody capture substance solid phase carrier. Further, it may contain a blocking solution, a reaction solution, a reaction stop solution, a reagent for treating the sample, and the like.

7. Immunological measurement of antibody by antibody capture method Antigen bound with the above-mentioned tag, labeled antibody against tag and anti-human IgG antibody, using a carrier immobilized with anti-human IgM antibody and / or anti-human IgA antibody, The target antibody can be measured.

For example, antibody measurement by ELISA by an antibody capture method using an enzyme as a labeling substance can be performed as follows, using a microplate to which an antibody against human IgM antibody is bound.

4. Antibody ELISA by antibody capture method (I) After diluting a serum-derived sample with a buffer, 5. Add to the antibody solid-phase plate prepared in step 2 and react at room temperature. After the reaction, washing with a buffer solution was performed. The mixture prepared by previously mixing the tag-bound antigen prepared in step 4 and the labeled anti-tag antibody prepared in step 4 and reacting them is added, and allowed to react at room temperature. After washing with a buffer solution, an enzyme substrate solution is added, the reaction is performed at room temperature, and then a reaction stop solution is added. The presence or absence of the antigen in the sample or the amount of the antigen is determined by measuring the absorbance using a colorimeter or the like.

4. IgM ELISA by antibody capture method (II) After diluting a serum-derived sample with a buffer solution, 5. Add to the antibody solid-phase plate prepared in step 2 and react at room temperature. After the reaction, washing with a buffer solution was performed. Add the tag-bound antigen solution prepared in
After reaction at room temperature and washing with a buffer solution, the labeled anti-tag antibody prepared in step 4 is added, followed by reaction at room temperature, washing with a buffer solution, addition of an enzyme substrate solution, reaction at room temperature, and addition of a reaction stop solution. The presence or absence of the amount of antigen in the sample or the amount of antigen is determined by measuring the absorbance using a colorimeter or the like.

Although the antibody in the sample can be measured by either of the methods (I) and (II), the tag-binding antigen and the labeled anti-tag antibody are premixed and reacted as in (I). In addition, when the mixture is added to the plate to which the antibody to be measured is bound, the number of steps and the time required for measuring the antibody are reduced. The immunological measurement of IgM by the antibody capture method using a fluorescent dye, a luminescent substance, and a radioisotope as a labeling substance can be similarly performed.

[0036]

[Examples] In the following, as an example of the present invention, one of the virus capsid antigen (VCA) group, which is a constituent component of EBV particles, among anti-EBV antibodies that are indicators of Epstein-Barr virus (EBV) infection. The following describes an example applied to an antibody capture method ELISA for detecting IgM antibody against p18 antigen having strong immunoreactivity. The present invention is not limited to the following examples.

[Example 1] Cloning of EBV p18 antigen gene 1. Preparation of genomic DNA (1) Culture of EBV genome-retaining cells Marmoset-derived cell line B95-8 that retains the EBV genome in the cells is RPMI 1640 medium containing 10% fetal calf serum (FCS) and 2 mM L-glutamine. The cells were cultured in 200 mL at 37 ° C. The culture medium was collected when the cell concentration reached about 2 × 10 ⁶ / mL.

(2) Preparation of genomic DNA The above culture was centrifuged at 160 × g for 10 minutes, and the supernatant was recovered. Add 20 (W / V)% polyethylene glycol, 1M to this supernatant.
An equal amount of a solution consisting of NaCl was added, mixed well, and then left overnight at 4 ° C. to precipitate EBV. After being left for 20 minutes at 13,000 × g
After centrifuging for minutes, the supernatant was discarded and the precipitate was dissolved in 1.8 mL of sterile ultrapure water. Then 0.1 M Tris-HCl (pH 7.8), 50 mM EDTA and 5% S
200 μL of DS buffer, followed by 20 mg / mL Protea
10 μL of seK (manufactured by Sigma) was added and reacted at 50 ° C. for 1 hour. After completion of the reaction, phenol / chloroform treatment was performed, followed by centrifugation at 10,000 xg for 5 minutes to recover the upper aqueous phase portion. The aqueous phase was precipitated with ethanol and recovered, and finally 200 μL of TE buffer (1 mM EDTA, 10 mM
It was dissolved in Tris-HCl (pH 7.5)) to obtain EBV genomic DNA.

2. Cloning of p18 antigen gene Using 1 μL of the EBV genomic DNA solution obtained in Example 1-1, Saiki
Gene Amp P according to the method [Nature, 324,163 (1986)].
The gene region containing the p18 antigen gene was amplified by the PCR method using CR Reagent Kit (Takara Shuzo). First, in order to incorporate it into an expression vector, two types of PCR oligonucleotide primers having the sequences of SEQ ID NOs: 1 and 2 in which a part of the original sequence of the p18 antigen gene was mutated and a restriction enzyme cleavage site was introduced were outsourced. (Espec Oligo Service). Next, 10 mM Tris-HCl (pH 8.3), 50 mM KC
EBV genome in a reaction solution containing 1,5 mM MgCl ₂ , 0.1% gelatin
1 μL DNA solution and 20 pmoles of each of the above two types of oligonucleotide primers and the Taq polyme attached to the above kit
rase 5 units were added to make a final volume of 50 μL.

This reaction solution was heated at 94 ° C. for 3 min. → [(94 ° C., 1.0 mi
n. → 50 ℃, 30sec. → 72 ℃, 2.0min.) × 25 cycles] → 72
The reaction was performed under the conditions of ℃, 7 min. As a result of fractionating the reaction-completed solution by 1.5% agarose gel electrophoresis, a DNA of about 570 bp PCR amplification product was confirmed. After collecting the PCR amplification product of this approximately 570 bp from within the gel with Geneclean II (manufactured by BIO 101),
Further, it was smoothed using DNA Blunting Kit (manufactured by Takara Shuzo).

Next, 2 μg of the cloning vector pUC19 (manufactured by Amersham Pharmacia Biotech) was digested with a restriction enzyme.
After digestion with 4 units of SmaI (manufactured by NEW ENGLAND BioLabs) at 30 ° C. for 2 hours, the 5 ′ end was dephosphorylated with alkaline phosphatase (manufactured by BRL).

After the above treatment, the PCR amplification product and pUC19 were treated with Liga.
Bonding using the Option Kit (Takara Shuzo), CaCl ₂ method [Mol
ecular Cloning, p250 Cold Spring Harbar Laborator
y] was introduced into E. coli JM109 (manufactured by Funakoshi).
Properly select 10 of the obtained transformants, purify the plasmid DNA by the alkali-SDS method of Birnboim et al. [Nuc. Acids Res., 7, 1513 (1979)], and have the PCR amplification product by restriction enzyme mapping. A clone (pp18) was obtained.

The plasmid DNA of the obtained clone was purified, and ABI PRISM ^™ 310 Ge manufactured by Perkin Elmer Japan Co., Ltd.
The base sequence of the DNA of the portion derived from the above PCR amplification product was determined with a netic Analyzer. As a result, it completely matched with the known base sequence (described in JP-A-6-90787), except for the portion mutated for the purpose of introducing into the expression vector.

[Example 2] Expression of fusion protein of GST and p18 antigen by E. coli 1. Preparation of expression vector 20 μg of the plasmid pp18 obtained in Example 1 was digested with the restriction enzyme Sal.
After digesting with 20 units of I (NEW ENGLAND BioLabs) at 37 ° C. for 2 hours, phenol extraction-ethanol precipitation was performed. DN
After dissolving A in sterile ultrapure water, the restriction enzyme BamHI (NEW ENGLAND B
(ioLabs) 20 units, digested at 37 ℃ for 2 hours, and then subjected to 1.5% agarose gel electrophoresis to generate a Geneclean II fragment of about 550 bp.
(Manufactured by BIO101).

Next, a GST fusion protein expression vector pGEX-5X-
5 μg of 1 (Amersham Pharmacia Biotech) was digested with the restriction enzyme SalI 20 units at 37 ° C. for 2 hours, and then phenol extraction-ethanol precipitation was performed. Dissolve DNA in sterile ultrapure water, digest with restriction enzyme BamHI 20 units at 37 ° C for 2 hours,
Perform 1% agarose gel electrophoresis, and analyze the fragment of about 4.9 Kbp with Ge.
Recovered with neclean II.

The above 550 bp fragment, 4.9 Kbp, was ligated and CaC
It was introduced into E. coli JM109 by the l ₂ method. After selecting 24 appropriately obtained transformants, the DNA was purified by the alkali-SDS method, and the p18 antigen was bound to the C-terminal side of GST by mapping with the restriction enzymes SalI and BamHI and determining the nucleotide sequence near the binding site. A clone (pGST-p18) having an expression vector encoding the fused protein (GST-p18) was obtained.

2. Expression of the fusion protein GST-p18 Expression vector pGST-p18 was used to transform E. coli BL21 (manufactured by Funakoshi) for protein expression to obtain transformed E. coli BL21 (pGST-
p18) was prepared. L-Broth medium (BIO101; 50 μg / mL Ampicillin (Nacalai Tesque) was added) to the transformant.
The cells were cultured at 37 ° C for 16 hours. Add 5 mL of this culture to new L-Bro
500 ml of th medium (containing 50 μg / mL Ampicillin) was inoculated.
After culturing at 37 ° C for about 3 hours, when OD600 reached 0.4-0.6, 0.1 mM Isopropyl-β-D-thiogalactopyranoside (IPT
G; manufactured by Nacalai Tesque, Inc.) was added, and the cells were further cultured for 3 hours. After the culture was completed, the cells were collected by centrifugation at 5,000 xg.

[0048] A part of the bacterial cells is a phosphate buffered saline (Ph
osphate Buffered-Saline (PBS)) and suspend the cells with an ultrasonic disruptor, then use the Laemmli method [Nature, 227,680].
(1970)] and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue (CBB). After electrophoresis, the anti-GST antibody (Amersham
Western blotting was performed using Pharmacia Biotech. As a result, in CBB staining, GST-p18
A band darker than other molecular weight bands was confirmed at a position of about 45,000, which is the predicted molecular weight of. As a result of Western blotting, this band reacted with the anti-GST antibody.
From the above, it was confirmed that the target protein was expressed.

3. Purification of fusion protein GST-p18 GST-p18 expressing bacterial cells recovered in Example 2-2 were microbial cell disruption buffer (20 mM Tris-HCl (pH8.0), 5 mM EDTA, 5 mM DTT, 50 mM Na
Cl) was suspended in 50 mL and sonicated. 13,000 crushed liquid
The supernatant was collected by centrifugation at × g. The supernatant was filtered with a 0.45 μm filter to obtain a sample solution. The sample solution was equilibrated with the cell disruption buffer and the amount of resin was 5 mL of Glutathio.
It was applied to a ne Sepharose column (GST Trap; Amersham Pharmacia Biotech) at a flow rate of 1 mL / min. After applying the total amount of the sample solution, the flow rate was increased to 2 mL / min, and the column was washed with the cell disruption buffer for 2 hours. After washing, an elution buffer containing 10 mM reduced glutathione added to the cell disruption buffer was sent to the column at 2 mL / min, and 3 mL of the protein elution fraction was collected. This fraction is SDS-PAGE
When separated by CBB staining, a single band with a molecular weight of about 45,000 was observed. As a result of analysis with Image Master (manufactured by Amersham Pharmacia Biotech Co., Ltd.) for assaying protein purity, it was confirmed to be almost 100%.

[Example 3] Detection of anti-EBV-IgM antibody using fusion protein expressed in E. coli 1. Preparation of anti-human IgM antibody solid-phase plate for antibody capture method ELISA Goat anti-human IgM monoclonal antibody (Miles) 50m
Dilute to a concentration of 15 μg / mL with M carbonate buffer (pH 9.5) and add 100 μL to polystyrene flat microplates (Nunc).
/ Dispensed in wells and left overnight at 4 ° C. The microplate that had been left standing for 18 hours or more was washed twice with 300 μL / well of Tris-HCl (pH 7.6) containing 0.05% Tween20 at the final concentration, and then Tris-HCl (pH 7. 6) to 20
An anti-human IgM monoclonal antibody solid phase microplate was prepared by adding 0 μL / well and allowing to stand at 4 ° C. overnight.

2. Preparation of enzyme-labeled anti-GST antibody Anti-GST polyclonal antibody solution (Amersham Pharmacia Biotech) 2.0mL (protein concentration 5mg / mL) 20m
It was dialyzed against 1 L of M carbonate buffer (pH 9.5) at 4 ° C. overnight. On the other hand, 1 mL of 4 mg of peroxidase (Type IV; Sigma)
Was dissolved in sterile ultrapure water, and then 0.2 mL of 0.1M sodium metaperiodate was added and mixed well. After standing for 20 minutes, it was dialyzed against 2 L of 1 mM acetate buffer (pH 4.0) at 4 ° C. overnight. After the dialysis, 1/3 volume of 20 mM carbonate buffer was added to the peroxidase solution to give an oxidized peroxidase solution. Then, the dialyzed anti-GST polyclonal antibody solution and the oxidized peroxidase solution were mixed and reacted at room temperature for 2 hours with stirring. 120 μL of a 4 mg / mL sodium borohydride solution was added to this mixed solution, and the mixture was stirred in ice water for 2 hours for reaction. After the reaction is completed, 10mM Na-phosphate buffer (pH 7.0) is used to separate the unreacted portion.
Using Sephacryl S-200 (Amersham Pharmacia
Gel filtration was performed with Biotech Co., Ltd. to obtain a purified fraction of peroxidase-labeled anti-GST antibody.

3. Detection of anti-EBV-IgM antibody (1) ELISA of IgM by antibody capture method (I) After removing the plate stock solution in the anti-human IgM monoclonal antibody solid-phase microplate well described in Example 3-1, the infectious monolayer was removed. 30 sera clinically diagnosed with nuclear disease and 50 normal sera
Specimen 200 with PBS (pH 7.0) containing 0.5% BSA and 0.05% Tween 20.
After doubling the dilution, 100 μL was added to the wells of the solid phase microplate, and the mixture was reacted at room temperature (15 ° C to 25 ° C) for 1 hour. After the reaction
The cells were washed 3 times with 200 μL / well of PBS (pH 7.0) containing 0.05% Tween20.

During the above reaction time, 0.5% BSA was added to the antigen solution.
And PBS containing 0.05% Tween 20 (pH 7.0) were used to prepare Example 2-3.
The purified product as described was diluted to 2 μg / mL and prepared. Next, the peroxidase-labeled anti-GST antibody described in Example 3-2 was added to 0.5% BSA.
And PBS (pH 7.0) containing 0.05% Tween 20 were diluted to 2 μg / mL to prepare a labeled antibody solution. The antigen solution and the labeled antibody solution were mixed in equal amounts to prepare an antigen-labeled antibody mixed solution, which was allowed to stand at room temperature for 10 minutes or more.

After reacting for 1 hour, the wells were washed 3 times with 200 μL / well of PBS (pH 7.0) containing 0.05% Tween20. Then,
Add 100 μL / well of antigen-labeled antibody mixture and incubate at room temperature for 1
Reacted for hours. After the reaction, PBS containing 0.05% Tween 20 (pH
7.0) Washed 5 times with 200 μL / well. After washing, 3.3 mg / mL
O-Phenylenediamine-containing 0.1M citric acid-phosphate buffer (pH 5.0), 0.02% hydrogen peroxide was added to the substrate solution 100
μL / well was added and reacted at room temperature for 30 minutes. After the reaction, 1.
The reaction was stopped by adding 100 μL / well of 5N sulfuric acid, and the main wavelength was 492 nm and the sub wavelength was 630 nm using a microplate colorimeter.
The OD value of each well was measured by. A sample with an OD value of cutoff or more was determined to be positive. Table 1 shows the performance of this ELISA when the clinical diagnosis was used as a control. The cut-off value was a value obtained by adding the standard absorbance three times the standard deviation to the average absorbance of normal human serum.

In this ELISA, the time required from the addition of the sample to the well to the measurement of the OD value with a colorimeter was about 3 hours. (2) IgM ELISA by antibody capture method (II) After removing the plate stock solution in the anti-human IgM monoclonal antibody solid phase microplate well described in Example 3-1,
The same serum used in (1) was diluted 200-fold with PBS (pH 7.0) containing 0.5% BSA and 0.05% Tween 20, and 100 μL was added to the wells of the solid-phase microplate at room temperature (15 ° C to 25 ° C). The reaction was carried out at (° C) for 1 hour. After the reaction, PBS containing 0.05% Tween 20 (pH 7.0)
Washed 3 times with 200 μL / well. Next, add 0.
It was prepared by diluting the purified product described in Example 2-3 to 1 μg / mL with PBS (pH 7.0) containing 5% BSA and 0.05% Tween20.

The antigen solution was added to the wells of the solid phase microplate at 100 μL / well, and then reacted at room temperature (15 ° C. to 25 ° C.) for 1 hour. After the reaction, PBS containing 0.05% Tween 20 (pH 7.
0) Washed 3 times with 200 μL / well. Then, the peroxidase-labeled anti-GST antibody described in Example 3-2 was added to 0.5% BSA and 0.05%.
A labeled antibody solution was prepared by diluting to 1 μg / mL with PBS containing Tween 20 (pH 7.0). Add 100 μL / well of this labeled antibody solution,
The reaction was carried out at room temperature for 1 hour. After reaction, contains 0.05% Tween 20
The plate was washed 5 times with 200 μL / well of PBS (pH 7.0). After washing 3.3
To a 0.1 M citric acid-phosphate buffer solution (pH 5.0) containing mg / mL O-phenylenediamine, 100 μL / well of a substrate solution prepared by adding 0.02% hydrogen peroxide solution was added and reacted at room temperature for 30 minutes. After the reaction, add 100 μL / well of 1.5N sulfuric acid to stop the reaction,
The OD value of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm using a colorimeter for microplates. A sample with an OD value of cutoff or more was determined to be positive. Table 1 shows the performance of this ELISA when the clinical diagnosis was used as a control. The cutoff value is the standard deviation of the average absorbance of normal human serum.
The value obtained by adding 3 times was used. In this ELISA, the time required from the addition of the sample to the well to the measurement of the OD value with a colorimeter was about 4 hours.

[0057]

[Table 1] * 1 Sensitivity (%) = Percentage of sera diagnosed as infectious mononucleosis by clinical diagnosis that were determined to be positive by ELISA * 2 Specificity (%) = Normal serum determined as negative by ELISA Serum percentage

From the above results, it is possible to detect the IgM antibody against EBV with high sensitivity and specificity by using the enzyme-labeled anti-GST antibody and the EBV antigen genetically fused with GST in the antibody capture method ELISA. Is possible. Furthermore, by premixing the above antibody and antigen,
It is possible to reduce the steps of A, which results in E
The time required for LISA can be shortened. Further, in the conventional construction of ELISA, the step of producing an antibody against EBV and the step of labeling the antibody with an enzyme label or the like can be omitted.

[0059]

INDUSTRIAL APPLICABILITY It is possible to detect an antibody with high sensitivity and high specificity by an immunological measurement method by an antibody capture method using an antibody against a labeled tag and an antigen bound with the tag. Further, in the conventional construction of an immunological measurement method, it became possible to omit the step of producing an antibody against an antigen and the step of labeling the antibody with an enzyme label or the like, which were necessary.

[0060]

[Sequence list]                                SEQUENCE LISTING <110> Denka Seiken <120> A method for immuno-detection of an antibody by an       antibody capture method <130> P01-0493 <140> <141> <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic <400> 1 ctatcagacg gatcctggca cgccggct 28 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic <400> 2 caccatgttt cgtggtcgac actgtttctt 30

[0061]

[Sequence list free text] SEQ ID NOs: 1 and 2: synthetic

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/569 G01N 33/569 L 33/58 33/58 Z // C12N 15/09 C12N 15/00 A (72) Inventor Toshinori Sato 1-2-2 Minamihonmachi, Gosen, Niigata Denka Seiken Co., Ltd. F-term (reference) 2G045 AA28 BA13 BB01 BB03 BB07 BB10 BB20 CA26 CB01 CB21 FB03 FB04 FB05 FB07 FB15 GC10 HA16 4B024A11 BA32 CA03 CA07 DA06 GA11 HA03

Claims (17)

[Claims] 1. A method for immunologically measuring an antibody against an antigen in a sample by an antibody capture method, which comprises using an antigen bound to at least a tag and a labeled antibody against the tag. 2. The antigen in the sample according to the antibody capture method according to claim 1, further comprising a plate on which a substance that binds to an antibody in the sample against the antigen bound to the tag is immobilized. Immunoassay method for antibody against sucrose. 3. (a) adding a sample to a plate on which a substance that binds to an antibody is immobilized, (b) adding an antigen having a tag bound to the plate, and (c) adding to the plate The immunological measurement method of the antibody with respect to the said antigen in a sample by the antibody capture method of Claim 1 or 2 including the process of adding the labeled antibody with respect to this tag. 4. The immunological measuring method according to claim 1, wherein the immunological measuring method is ELISA. 5. The immunochemical measurement of an antibody by the antibody capture method according to claim 1, wherein the antigen bound with the tag and the antibody against the labeled tag are premixed before use. Method 6. The antibody capture according to any one of claims 1 to 5, wherein the antigen bound with the tag is a fusion protein of the tag and the antigen produced by a gene recombination method. Immunoassay method for antibody 7. The tag is His ₆ , GST (Glutathione S-Tran
sferase), MBP (Maltose Binding Protein), galactose-binding protein, and thioredoxin. The immunology of the antibody by the antibody capture method according to any one of claims 1 to 6, which is characterized by comprising a protein selected from the group consisting of: Measurement method 8. The method for immunologically measuring an antibody by the antibody capturing method according to claim 1, wherein the antigen is an Epstein-Barr virus (EBV) antigen. 9. The method according to claim 1, wherein the substance that binds to the antibody in the sample is an anti-human immunoglobulin antibody.
9. A method for immunologically measuring an antibody against the antigen in a sample by the antibody capture method according to any one of 8 above. 10. The substance that binds to the antibody in the specimen is anti-human
The method for immunologically measuring an antibody by the method for capturing an antibody in a specimen according to claim 9, which is an IgM antibody. 11. A reagent for measuring an antibody in a sample by an antibody capture method, which comprises at least a tag-bound antigen and an antibody against the labeled tag. 12. The antibody measuring reagent according to claim 11, further comprising a plate on which a substance that binds to an antibody in a sample for an antigen to which a tag is bound is immobilized. 13. The antibody measuring reagent according to claim 11 or 12, wherein the antigen bound with the tag is a fusion protein of the tag and the antigen, which is produced by a gene recombination method. 14. The tag is His ₆ , GST (Glutathione S-Tr
ansferase), MBP (Maltose Binding Protein), a galactose binding protein, and a protein selected from the group consisting of thioredoxin.
13. The antibody measuring reagent according to any one of 13 above. 15. The method according to claim 1, wherein the substance that binds to the antibody in the sample is an anti-human immunoglobulin antibody.
The antibody measuring reagent according to any one of 1 to 14. 16. A substance that binds to an antibody in a sample is anti-human
The antibody measuring reagent according to claim 15, which is an IgM antibody. 17. The method according to claims 11 to 1, characterized in that the antigen is the Epstein-Barr virus (EBV) antigen.
6. The antibody measuring reagent according to any one of 6 above.