JPH0382962A - Method of detecting antibody to hepatitis b virus core antigen - Google Patents

Abstract

PURPOSE:To detect an anti-HBc antibody with high sensitivity by using a hepatitis B virus core antigen having a label bonded thereto or a substance (HBc antigen) having antigenicity the same as that of said antigen. CONSTITUTION:At first, an HBc antigen is immobilized on a proper solid phase and, next, the prepared solid phase is brought into contact with a specimen containing the HBc antigen. Subsequently, the solid phase is separated from a liquid phase. Then, an HBc antigen having a label bonded thereto is brought into contact with the previously obtained solid phase. Next, the solid phase is separated from the liquid phase and the label in the obtained solid or liquid phase is detected. Or, the HBc antigen is immobilized on a proper solid phase. Next, the prepared solid phase, a specimen containing the antibody to a hepatitis B virus core antigen and a hepatitis B virus core antigen having a label bonded thereto or a substance having antigenicity same to that of the labeled antigen are brought into contact with each other. Subsequently, the solid phase and the liquid phase are separated and the label in the obtained solid or liquid phase is detected. By this method, an HBc antibody can be detected with high sensitivity.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is directed to antibodies against hepatitis B virus Core antigen or a substance having antigenicity equivalent to this (hereinafter simply referred to as HBc antigen) (hereinafter simply referred to as anti-HBc antibody). related to the measurement method of More specifically, the present invention relates to a method for measuring anti-HBc antibodies by immunoassay using HBc antigen bound to a label.

(Prior art) Serological tests for hepatitis B virus (hereinafter referred to as HBV) are mainly typified by detection of HBV surface antigen (hereinafter referred to as HBs antigen) and antibodies against it (hereinafter referred to as anti-HBs antibody). It's here.

However, during transient infection, HBs antigen becomes negative,
Furthermore, diagnosis of hepatitis B during the period when HBs antibodies are not detected (core window), differential diagnosis between the onset of initial infection and onset from persistent infection carriers, screening of so-called high-risk groups centered on medical workers, and HBs antigen Anti-HBc antibody tests are of great significance, such as in serological diagnosis at the time of vaccination. Anti-HBc antibody testing has traditionally been performed using immunoadhesive hemagglutination method,
Passive hemagglutination method, radioimmunoassay (hereinafter referred to as RIA)
), enzyme immunoassay (hereinafter referred to as E-4A), etc. - RIA and EIA, which have high sensitivity in membranes, can be roughly divided into competitive methods and Sand-Deutsch methods based on their measurement principles. The competitive method in antibody testing involves competitively reacting a certain amount of labeled antibody with the antibody to be measured against a certain amount of antigen, and the amount of labeled antibody that binds to the antigen varies depending on the amount of antibody to be measured. Therefore, by measuring the amount of bound or unbound labeled antibody, the amount of antibody to be measured can be determined.

On the other hand, for example, in the Sand-Deutsch method, antibodies in a sample are trapped in an antigen insolubilized on a solid phase, and labeled anti-immunoglobulin antibodies (hereinafter referred to as labeled antibodies) are labeled after or without removing antibodies that do not bind to the solid phase. The antibody concentration in the sample can be determined by reacting two antibodies (individual antibodies) and measuring the labeled second antibody that bound to the antibody trapped on the solid phase, or the labeled second antibody that did not bind. . In such measurements, it is desirable that the sensitivity be sensitive.

Factors that affect sensitivity include the detection sensitivity of the label and the affinity between antigen and antibody, but it also depends on the measurement method described above. That is, in the competitive method, the measurement sensitivity does not decrease unless the amount of labeled antibody is reduced, but the amount of label measured thereby decreases. Therefore, there is a certain limit to improving the measurement sensitivity due to the detection sensitivity of the label.

On the other hand, the Sand-Deutsche method, which uses an excess of labeled secondary antibody, tends to have a high background due to nonspecific adsorption of immunoglobulin in the sample to the solid phase, but the competitive method uses a fixed amount of labeled antibody. The measurement sensitivity tends to be higher than that of .

(Problems to be Solved by the Invention) As mentioned above, although it is generally easier to improve the measurement sensitivity of the Sand-Deutsch method compared to the competitive method, the competitive method has not been adopted for the detection of anti-HBc antibodies in the past. Detection by the Sanderutsch method is not known.

(Means for Solving the Problems) The present invention is a method for detecting anti-HBc antibodies by the Sand-Deutsch method, thereby making it possible to detect anti-HBc antibodies with improved measurement sensitivity. That is, the present invention is a method for detecting an anti-HBc antibody using a label-bound HBc antigen or a substance having antigenicity equivalent thereto. Specifically, the present invention includes two steps or one step consisting of the following operations.
It includes the step sandwich ml+standard method.

(A) Immobilization of hepatitis B virus Core antigen or a substance with antigenicity equivalent to this onto a suitable solid phase; (B) Immobilization of the solid phase prepared in (A) and hepatitis B virus C.
(C) Separating the solid phase and liquid phase; (D) Hepatitis B virus Core antigen bound to a label or a substance with equivalent antigenicity. (C) contacting the solid phase obtained in (C), (E) separating the solid phase and liquid phase, (P) detecting the label in the solid phase or liquid phase obtained in (E). or (a) an operation of immobilizing hepatitis B virus Core antigen or a substance having antigenicity equivalent to this on a suitable solid phase; (b) a solid phase prepared in (a); hepatitis B virus C
an operation of contacting a sample containing an antibody against the ore antigen with a hepatitis B virus core antigen to which a labeled substance has been bound or a substance having antigenicity equivalent to this, (C) an operation of separating a solid phase and a liquid phase, (c+ ) This is a detection method consisting of an operation of detecting a labeled substance in the solid phase or liquid phase obtained in (c).

Furthermore, when the anti-HBc antibody belongs to immunoglobulin M (hereinafter referred to as IgM), the present invention particularly includes a two-step or one-step sandwich assay method comprising the following operations.

(A) Immobilizing the anti-IgM antibody on a suitable solid phase, (
I3) Solid phase prepared in (A) and hepatitis B virus C
(C) Separation of the solid phase and liquid phase; (D) Hepatitis B virus Core antigen bound to a labeled substance or antigenicity equivalent thereto. (C) contacting the substance with the solid phase obtained in (C), (E) separating the solid phase and liquid phase, (P) detecting the labeled substance in the solid phase or liquid phase obtained in (E). (a) Immobilizing the anti-1gM antibody on a suitable solid phase, (
b) Solid phase prepared in (a) and hepatitis B virus Co
an operation in which a sample containing an IgM type antibody against re antigen is brought into contact with a hepatitis B virus C0re antigen to which a labeled substance is bound or a substance having antigenicity equivalent to this; (C) an operation to separate a solid phase and a liquid phase; (d) A detection method consisting of an operation for detecting a labeled substance in the solid phase or liquid phase obtained in (e). The present invention will be explained in detail below.

HBc antigens include naturally occurring forms of H, such as those artificially produced by genetic recombination technology, or antigens extracted and purified from viruses or virus-infected tissues.
In addition to the Bc antigen, for example, a peptide containing the antigenic determinant portion of the antigen and having antigenicity equivalent to that of the HBc antigen may be used.

Labels that bind to HBc antigens include radioactive isotopes used in normal RIA methods, enzymes used in normal EIA methods, and fluorescent, color-forming, light-absorbing substances, total refraction, etc. Just use it. In particular, it is preferable to use enzymes such as alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, etc. from the viewpoint of stability of the reagent and absence of risk of exposure to radiation. Binding of these labels to the HBc antigen is achieved by following commonly known conjugate preparation methods, but it can also be prepared, for example, by the methods described in the Examples of the present invention.

There are no particular limitations on the solid phase on which the HBc antigen or anti-1gM antibody is immobilized, but examples include polystyrene, polyethylene, polyvinyl chloride, latex, agarose, cellulose, methacrylate glass, and the like. There is no particular restriction on its shape, such as bead-like, sheet-like, stick-like, tube-like, or plate-like. For example, it is also possible to make a cup or a microplate using the above-mentioned materials and use the inner wall as a solid phase.

Immobilization of the HBc antigen or anti-1gM antibody onto the solid phase can be achieved, for example, by commonly known adsorption methods or chemical bonding methods. Here, if the solid phase is made of a material that has the property of adsorbing various proteins, after the immobilization,
It is preferable to perform a blocking operation using BSA (bovine serum albumin) or the like.

The contact between the solid phase and the sample may be carried out under conditions that allow an immunoreaction between the anti-HBc antibody in the sample and the HBc antigen or anti-IgM antibody immobilized on the solid phase. good.

Separation of solid phase and liquid phase is described, for example, in JP-A No. 62-273453.
No. 63-1.2959, etc. can be used.

2. The anti-HBc antibody in the sample can be detected by detecting the labeled substance in the solid phase separated by the above separation operation. Furthermore, when carrying out the present invention, a certain amount (a known amount) of
When using HBc antigen bound to a labeled substance, anti-HBc antigen in the sample can also be detected by detecting the labeled substance in the liquid phase.
Antibodies can be detected. The detection imitation of the marked object may be carried out based on the special properties of the marked object used, for example, in several cases.
This is accomplished by methods such as line detection, fluorescence detection, absorbance detection, spectral detection, and atomic absorption. When an enzyme is used as a label, a substrate that generates a signal detectable by the above-mentioned method upon the action of the enzyme may be added, and then a detection operation may be performed.

For example, if the enzyme is horseradish peroxidase, 2
, 2'-azinobis-[3-ethylbenzthiazolinesulfonic acid] ammonium salt-H202 and 0-phenylenediamine can be exemplified as substrates. For example, enzyme β
Examples of -D-galactosidase include 0-nitrophenyl-β-D galactopyranoside. For example, if the enzyme is alkaline phosphatase, examples include p-nitrophenyl phosphate and 4-methylumbelliferyl phosphate.

(Effects of the Invention) According to the present invention, anti-HBc antibodies can be measured by the Sand-Deutsch method, and anti-HBc antibodies can be detected with higher sensitivity than measurements by conventional competitive methods. Therefore, by investigating the amount of labeled substance detected when carrying out the present invention on a sample containing a known concentration of anti-HBc antibody, it is also possible to quantify the unknown concentration of anti-HBc antibody in the sample. It is.

(Examples) Examples will be described below to explain the present invention in more detail, but these are merely examples and do not limit the present invention.

Example 1; Two-step measurement of anti-HBc antigen (1) Immobilization of HBc antigen HBc antigen obtained by genetic recombination technology was
Phosphate saline buffered relative (
(hereinafter referred to as PBS) and ethylene-vinyl acetate copolymer beads with a diameter of 1.4 to 1.5 mm at room temperature.
Shake at 127 rpm for an hour and a half. After removing the antigen solution, PBS containing 0.5% bovine blood 111 albumin
(hereinafter referred to as PBS-BSA) was added and allowed to stand for 3 hours to perform a blocking operation.

Regarding the preparation of HBc antigen, please refer to Nature No. 2.
This was done with reference to Vol. 82, p. 575 (1979).

(2) ALPase labeling of HBc antigen A dioxane solution of anhydrous S-acetylmercaptoconophosphoric acid was added to the HBc antigen to obtain S-acetylmercaptosuccinylated HBc antigen.

An ethanol solution of N-succinimidyl-3-(2-pyridyldithio)propionate was added to alkaline phosphatase (ALPase) to obtain pyridyl disulfidated ALPase. The S-acetylmercaptosuccinylated HBc antigen 5 was converted into a mercaptosuccinylated HBc antigen using hydroxylamine, and the pyridyldisulfidated ALPas
The mixture was mixed with solution e at a weight ratio of 1=6, and allowed to stand at 4° C. for 4 days. Unreacted ALPase was removed using a molecular sieve chromatograph (manufactured by Tosoh ■, trade name G3000SW) to obtain a conjugate.

(3) Two-step measurement of anti-HBc antibody Place 12 beads immobilized with HBc antigen in a measuring cup, add 100 μl of phosphate buffer (pH 6.6) containing 0.5% bovine serum albumin, and sample. 50 μl was added and stirred at 37° C. for 20 minutes. 0.1% Tween 2
0 (Tween20) (hereinafter PBS-
After washing three times with Tween, the conjugate was
00 μm was added and stirred at 37° C. for 20 minutes. To remove unreacted conjugate, P B S -T w
After washing three times with e en, 100 μl of 1 mM 4-methylumbelliferyl phosphate solution (pH 10,0) was added, and the mixture was stirred at 37° C. for 10 minutes. Next, 500 μA of phosphate buffer containing 1M ethyl-6-diaminetetraacetic acid was added to stop the reaction, and the fluorescence intensity of the nucleic acid was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm. The results are shown in Figure 1.

In addition, the samples used in the present invention were rabbit antiserum (manufactured by Cambridge Medical Technology) against the human hepatitis B virus core antigen, with stock solutions of 100, 10, 53.3, 2, and 1.4.1, respectively. (xl
O-4) times and control (OXIO').

The results show that the detected fluorescence intensity is correlated with the anti-HBc antibody concentration in the sample.

Example 2: One-step measurement of anti-HBc antibody Twelve beads with immobilized HBc antigen prepared in the same manner as in Example 1 were placed in a measuring cup, and a sample of 50 μm was placed.
and conjugate 10 prepared in the same manner as in Example 1.
After adding 0 μA, the mixture was stirred at 37° C. for 40 minutes. PBS-Twee to remove unreacted conjugate.
After washing three times with n, add 100 μl of 1 mM 4-methylumbelliferyl phosphate solution (pH 10,0) and incubate at 37°C.
The mixture was stirred for 10 minutes. Next, 500 μl of IM phosphate buffer containing ethylenediaminetetraacetic acid was added to stop the reaction, and the fluorescence intensity of the nucleic acid was measured at an excitation wavelength of 365 nm.
Measurement was performed at a fluorescence wavelength of 450 nm.

The results are shown in Figure 2.

The samples used in the present invention were rabbit anti-blood/1lJ (manufactured by Cambridge Medical Technology Co., Ltd.) against human hepatitis B virus core antigen, and the stock solutions were 100, 10, 5°, 3.3, 2, and 1, respectively. .4.1
(XIO-4) times and control (OXIO-')
It is.

The results show that the detected fluorescence intensity is correlated with the anti-HBc antibody concentration in the sample.

[Brief explanation of drawings]

FIG. 1 shows the results of Example 1 of the present invention, and FIG. 2 shows the results of Example 2 of the present invention. In the figure, the horizontal axis is the dilution rate (XIO
-'), and the vertical axis is the fluorescence intensity (arbitrary unit ) is shown.

Claims (6)

[Claims] (1) A method for detecting antibodies against hepatitis B virus core antigen using hepatitis B virus core antigen bound to a labeled substance or a substance with equivalent antigenicity. (2) Claim No. 1, characterized in that it consists of the following operations (
The method described in section 1). (A) Immobilization of hepatitis B virus Core antigen or a substance with equivalent antigenicity on a suitable solid phase; (B) The solid phase prepared in (A) and hepatitis B virus Co.
(C) Separating the solid phase and liquid phase; (D) Hepatitis B virus Core antigen to which a label is bound or a substance with equivalent antigenicity. (C) contacting the solid phase obtained in (C), (E) separating the solid phase and liquid phase, (F) detecting the labeled substance in the solid phase or liquid phase obtained in (E). or (a) Immobilizing the hepatitis B virus Core antigen or a substance with antigenicity equivalent to this on a suitable solid phase; (b) the solid phase prepared in (a) and the hepatitis B virus. Co
(c) separation of solid phase and liquid phase; (d) ) An operation for detecting the labeled substance in the solid phase or liquid phase obtained in (c). (3) Claim No. 1, characterized in that it consists of the following operations (
The method described in section 1). (A) Immobilizing the anti-immunoglobulin M antibody on a suitable solid phase; (B) the solid phase prepared in (A) and hepatitis B virus Co.
(C) Separating the solid phase and liquid phase; (D) Hepatitis B virus Core antigen bound to a label or an antigen equivalent thereto; (C) An operation of bringing the solid phase obtained in (C) into contact with a substance having a property, (E) An operation of separating the solid phase and liquid phase, (F) A labeled substance in the solid phase or liquid phase obtained in (E) or (a) Immobilizing the anti-immunoglobulin M antibody on a suitable solid phase, (b) Immobilizing the solid phase prepared in (a) and hepatitis B virus Co.
(c) separating a solid phase and a liquid phase; (c) contacting a sample containing an immunoglobulin type M antibody against re antigen with a hepatitis B virus core antigen bound to a labeled substance or a substance having antigenicity equivalent thereto; (c) separating a solid phase and a liquid phase; (d) An operation for detecting the labeled substance in the solid phase or liquid phase obtained in (c). (4) The method according to claim (1), wherein the hepatitis B virus Core antigen or a substance having antigenicity equivalent to this is artificially produced by genetic recombination technology. . (5) Claims (1) to 1, wherein the labeled substance is an enzyme, a radioisotope, or an optically detectable compound.
The method described in any of paragraph (3). (6) Claim No. (2) or (3) wherein the labeled substance is an enzyme and the operation for detecting it consists of an operation of adding a substrate of the enzyme and an operation of detecting a reaction product subjected to the action of the enzyme. The method described in section.