JP2587969B2 - Virus antibody detection reagent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a reagent for detecting an acquired immunodeficiency syndrome-associated virus antibody, and more particularly to detection of an acquired immunodeficiency syndrome-associated virus antibody using a recombinant HIV antigen. For indirect agglutination reaction reagents.

[Related Art] Acquired Immunodeficiency Syndrome
Syndrome (commonly called AIDS) is a new disease first recognized in 1981. [MSGottilb et a
l., N. Eng, J. Med., 305, 1425 (1981)]. Later, in France, lymph node disease syndrome (Lymphadenopathy Syndrom
e) A retrovirus identified as LAV by the patient is [F.
rrs-Sinoussi et al., Science, 220, 868 (1983)],
And in the United States, AILV patients and pre-AIDS patients
−III [M. Popovic et al., Science, 224, 497 (1984)]
And ARV [JA Levy et al., Science, 225, 840 (198
4)] were discovered one after another. Recently, the LAV, HTLV-III, and ARV genes have been completely sequenced and their retroviruses have been shown to be mutants of the same virus [L. Ratner et al., Nature. , 313, 277 (1985), R. Sa
nchez Pescaoor et al., Science, 227, 484 (1985), S.
Wain Hobson et al., Cell, 40, (1985)].

This AIDS-causing virus is generally HIV (Human Immunod
HIV is commonly referred to as eficiency virus), but it is transmitted and infected by human-to-human contact and blood transfusion. On this occasion,
It is also known that antibodies reacting specifically with AIDS-related viruses occur in the blood of infected humans. Therefore, if the presence or absence of HIV infection is detected by detecting this antibody, it is considered that this will lead to the prevention of AIDS.

Accordingly, the present inventors have previously proposed an indirect agglutination reagent for detecting an AIDS-related virus antibody comprising carrier particles sensitized with an HIV antigen component (see JP-A-62-182662).

On the other hand, HIV has RNA as a nucleus, and is composed of a nuclear protein (core protein) that wraps the RNA and a reverse transcriptase, and an outer glycoprotein (env protein) that further wraps the nucleus.

HIV infection produces antibodies against the constituent proteins of these viruses in vivo.According to previous studies, there are so-called carrier groups that have been infected but have not developed HIV and those that have developed AIDS. Some reports have shown that differences were found in the amount of antibody against each of the core protein represented by p24 and the env protein represented by gp41. HIV infection usually produces antibodies against both the core protein and the env protein.However, after a certain period of time after infection, a difference in the amount of their production occurs and the antibody titer against the core protein begins to decrease. From ARC (AIDS rela
It has been reported that the symptoms of bed couplex begin to shift to AIDS (Joepet al., British Med. J.,
1986).

Therefore, anti-HIV-core (p24) anti-HIV antibody and anti-HIV
The detection of -env (gp41 or p41) protein antibody is of great clinical significance in tracking the transition from HIV infection to ARC and AIDS through carriers.

Conventionally, these anti-HIV-core (p24) protein antibodies, HI
It has been proposed to measure V-env (gp41 or p41) protein antibody by Western blotting or EIA [LANCET, Nov. 29, 1986, December 12, 1986].
See pages 33 to 1237]. However, in the former Western blot method, the HIV antigen protein component is transferred to a nitrocellulose membrane after SDS electrophoresis, and this membrane is reacted with the test serum by the EIA or RIA method, but the operation is complicated. The disadvantage is that it takes more than two days for the results to be obtained. Also, in the latter EIA method (Abbot's method), the reaction with the sample requires ten and several hours, the number of reaction steps is large, and the method is complicated.

[Problems to be Solved by the Invention] The present invention overcomes the above-mentioned drawbacks of the conventional method, and provides a simple and rapid anti-HIV-core (p24) protein antibody and anti-HIV-env.
(P41) It is an object of the present invention to provide a detection reagent capable of detecting a protein antibody, and the present inventors have conducted intensive studies for this purpose.
Using carrier particles sensitized with e (p24) protein and / or HIV-env (p41) protein under specific pH conditions,
Simple and quick anti-HIV-c
The inventors have found that an ore (p24) protein antibody and an anti-HIV-env (p41) protein antibody can be detected, and have completed the present invention.

[Means for Solving the Problems] However, according to the present invention, HIV-p24 and / or HIV
-An indirect agglutination reagent for detecting an acquired immunodeficiency syndrome (AIDS) -related virus antibody characterized by comprising gelatin particles sensitized to p41 at p7.3 to 8.5.

HIV-p24 is a kind of core protein with a molecular weight of about 2400 surrounding RNA present in the nucleus of HIV, and HIV-p41 is HIV-p41.
V is a component obtained by removing sugar from gp-41 (a molecular weight of about 4100), which is a kind of glycoprotein constituting the envelope of V. All of these proteins are known. For example, ANALYTICAL BIOCHE
According to the method described in MISTRY 1613 , 370-379 (1987),
HIV-p24 and HIV-p41 can be obtained by a gene recombination method using a bacterium such as Escherichia coli. These HIV-p24 and HI
V-p41 may be used alone or in combination.

Gelatin particles for sensitization with the above-mentioned HIV-p24 and / or HIV-p41 (Japanese Patent Application Laid-Open No.
No. 58) has excellent advantages as a carrier for an indirect agglutination reaction for detecting AIDS-related virus antibodies, for example, because it has few nonspecific reactions and can add properties depending on the application.

Sensitization of gelatin particles with HIV-p24 and / or HIV-p41 can be carried out using a method known per se, for example, tannic acid, glutaraldehyde, bisdiadibenzidine, tolylene diisocyanate, difluoro It can be performed by a method of chemically bonding or physically adsorbing using a so-called coupling agent such as dinitrobenzene, carbodiimides, quinones, and chromium chloride.

Sensitization may be performed under slightly alkaline conditions, and pH 7.5 to
The reaction is preferably performed within the range of 8.0. When sensitization is performed outside this pH range, a reagent excellent in detection sensitivity is generally not obtained. For example, HIV-gp24 or HIV-gp24 as a reference was added to gelatin particles under various pH conditions by the method described in Example 1 or 2 described below.
The results of testing the sensitivity when sensitizing gp41 are shown in Table 1 below.

Table 1 Sensitization pH control test Potency test 6.0 − − 7.0 − + 7.5 − + 8.0 − + 9.0 − + 10.0 + Unmeasurable In Table 1 above, -indicates that no agglutination reaction occurred, and + indicates Indicates that an agglutination reaction has occurred. Also,
The control test is a test using a serum diluted solution containing no AIDS-related virus antibody as a specimen, and a positive result in this test is a false reaction. On the other hand, the titer test
HIV-env (p41) protein antibody or anti-HIV-core (p24)
This is a test in which a serum solution containing a protein antibody is used as a specimen, and the larger the number, the higher the sensitivity.

HIV-p24 and / or HIV obtained as described above
The gelatin particles sensitized with -p41 are usually stored in a lyophilized state, restored after use, and subjected to an indirect agglutination reaction.

Detection of an AIDS-related virus antibody by indirect agglutination using the sensitized gelatin particles of the present invention can be performed in exactly the same manner as the indirect agglutination reaction known per se, for example, by placing a test serum in a well of a microplate. The dilution line is formed, and the sensitized gelatin particles of the present invention are added to each well and mixed, and the mixture is allowed to stand usually for about 1 to 2 hours. The aggregation image of the sensitized gelatin particles is observed to determine the presence or absence of aggregation. This is done by:

More specifically, the sensitized gelatin particle suspension was
An equal volume of a two-fold serial dilution of DS patient serum (sample) is added to the wells of a 96-well microplate, mixed, and allowed to stand at room temperature. After 1 to 3 hours, when observing each hole, the sensitized gelatin particles spread all over the bottom in the hole with a low dilution factor, and an aggregated image was observed. I can't. On the other hand, when unsensitized gelatin particles were used, no aggregation image was observed in any of the low- to high-diluted serum. In addition, when the HIV antibody negative human serum was examined in the same manner, no aggregated image was observed when either the sensitized gelatin particles or the unsensitized gelatin particles were used.

This allows HIV-p on the sensitized gelatin particles in the sample.
24 or an HIV antibody that specifically reacts with HIV-gp41 or p41 [anti-HIV-core (p24) protein antibody, anti-HIV-env (gp
41 or p41) protein antibody etc.] can be confirmed.

The sensitized gelatin particles of the present invention may be used as a reagent for detecting an AIDS-related virus antibody, as usual, with unsensitized gelatin particles, standard serum (positive serum for control), a sample diluting solution, and A kit can be put into practical use by combining it with a solution for dissolving a lyophilized product of sensitized gelatin particles.

In the present invention, p41 is used for convenience with respect to gp41, but more accurately refers to a protein in which sugar is removed from gp41.

[Examples] Next, the methods for preparing the sensitized gelatin particles of the present invention and the indirect agglutination test using the same will be described more specifically with reference to the following examples.

Reference Example 1 (1) Preparation of HIV-env (gp41) Protein Antigen Sensitized Gelatin Particles Gelatin particles produced according to the method described in Example 1 of JP-A-58-113754 were adjusted to pH 7 containing 5 ppm of tannic acid. .2 0.15M phosphate buffered saline (0.15MP
BS (abbreviated as pH 7.2)) so as to have a particle concentration of 2.5%, and heated at 37 ° C for 10 minutes. The particles were collected by centrifugation, washed three times with a physiological saline solution by a centrifugation method, and then dispersed in 0.15 M PBS (pH 8.0) so as to be 5%.

HIV-env (gp41) protein antigen was added to 0.15 M PBS (pH 8.
0), the mixture was diluted to 20 μg / ml with the above-mentioned tannic acid-treated particle dispersion, and an equal volume of this diluent was mixed.
After heating for 1 minute, the particles were sensitized with the HIV-env (gp41) protein antigen. The sensitized gelatin particles are centrifugally washed three times with a physiological saline solution, and then suspended in a suspension (disodium hydrogen phosphate.
1.920 g of dodecahydrate, 0.291 g of potassium dihydrogen phosphate, 0.438 g of sodium chloride, 0.25 g of gum arabic, 1.5 g of glycine, 0.1 g of sodium azide and 1.0 ml of normal rabbit serum in a total volume of 100 ml
(A solution dissolved in purified water). This dispersion was freeze-dried to obtain a freeze-dried product of HIV-env (gp41) protein antigen-sensitized gelatin particles.

(2) Preparation of test serum dilution solution 1.20 g of disodium hydrogen phosphate / 12 hydrate, 0.291 g of potassium dihydrogen phosphate, 0.438 g of sodium chloride, 0.25 g of gum arabic, 0.15 g of sodium azide To obtain a serum diluted solution.

(3) Preparation of kit In addition to the freeze-dried product of the sensitized gelatin particles, a freeze-dried product of the antigen-unsensitized gelatin particles and a freeze-dried product of the control positive serum were separately prepared. The test serum dilution solution was combined to prepare a test kit for detecting anti-HIV-env (p41) protein antibody.

Example 1 (1) Preparation of HIV-core (p24) Protein Antigen-Sensitized Gelatin Particles Instead of the HIV-env (gp41) protein antigen used in Example 1, HIV-core (p24) protein antigen (Centcore) And repeated the same operation to obtain HIV-cor
e (p24) protein antibody antigen-sensitized gelatin particles were prepared.

(2) Preparation of Kit An HIV-core (p24) protein antibody detection reagent kit was prepared using the same reagents as in Example 1.

Example 2 Using the test kits obtained in Examples 1 and 2, indirect agglutination tests were performed on various test sera.

 The results obtained are shown in Table 2 below.

From the results shown in Table 2 above, the HIV-env according to the present invention
(Gp41) Protein antigen-sensitized gelatin particles and HIV-core
(P24) Indirect agglutination using protein antigen-sensitized gelatin particles was performed using anti-HIV-env (gp41) protein antibody and anti-HIV
It can be seen that the detection of the -core (p24) protein antibody has the same detection sensitivity and specificity as the Western blot method.

[Effects of the Invention] By using the reagent of the present invention, a large number of samples can be quickly measured by a simple operation by an indirect agglutination reaction. For example, in the case of the fluorescent antibody method or the enzyme antibody method, washing and additional operations are required until the result is obtained from the start of the test, but when the reagent of the present invention is used, it is only necessary to add to the diluted serum, It is very convenient to leave it still until results are obtained. In addition, the result can be determined with the naked eye without any special equipment. These points are
Suitable for testing a large number of samples.

If an AIDS-related virus antibody is detected, it is considered that the virus has the AIDS-related virus in the body. Is very clinically useful because it can prevent

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Claims (1)

(57) [Claims] 1. An indirect agglutination reagent for detecting an acquired immunodeficiency syndrome-associated virus antibody, comprising gelatin particles sensitized with HIV nuclear protein P24 and / or HIV outer protein p41 at pH 7.5 to 8.0. .