JP2852656B2 - Method for producing human immunodeficiency virus type 2 antigen and reagent for detecting the antibody using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] Acquired Immunodeficiency Syndrome (hereinafter, “AID”)
S) is the first reported disease in 1981. Subsequent research has identified the causative virus of AIDS and has identified the human immunodeficiency virus (Human Immunodeficiency V).
irus (hereinafter referred to as "HIV"). HIV is transmitted by sexual contact between humans and blood transfusions, and develops AIDS after a latent period. For the prevention of AIDS, various reagents for detecting a specific antibody against HIV have been developed and used for screening of blood donation and the like.

Recently, however, a new human retrovirus distinct from HIV has been isolated from AIDS patients in West Africa (F.
Clovel et al, Science, 233, 343 (1981)] Human Immunodeficiency Virus type 2,
(Hereinafter referred to as "HIV-2") [F. Clovel et
al, Nature, 324, 691 (1986)]. The nucleic acid sequence of the HIV-2 gene was also completely analyzed, and was shown to differ from the HIV nucleic acid sequence [M. Guyoder et al, Nature, 326, 662 (19).
87)]. Furthermore, there was a difference in the molecular weight of the protein also at the protein level, and it was found that the serological cross-reactivity was limited to only the core protein having a molecular weight of 24,000, and in particular, the envelope glycoprotein did not show any cross-reactivity (F .Clovel et al, Nature,
324,691 (1986)].

For this reason, a reagent that detects only a specific antibody against HIV cannot detect a specific antibody against HIV-2, which is the other causative factor of AIDS, and thus requires a reagent that can detect a specific antibody against HIV-2.

The present invention relates to a method for producing an antigen of HIV-2, which is another causative factor of AIDS, and a reagent used for detecting a specific antibody against HIV-2 using the antigen.

[Conventional technology]

HIV-2 is isolated, and a continuous producer cell is immediately established to obtain HIV-2 antigen.
An enzyme-linked immunosorbent assay, western blot, for detecting specific antibodies to -2 has been developed.

As a persistently infected cell line of HIV-2, HUT-78 and CEM which are human cancerous T cell lines are infected with HIV-2, and HIV-2 is infected.
Has been reported to be a persistently infected cell line [F. Clov
el et al, Science, 233, 343 (1986)].

The above-mentioned HIV-2 persistently infected cell line is cultured, and the obtained purified HIV-2 is treated with "0.1% Triton X-100" to obtain HIV-
It has been reported that two antigens were obtained and enzyme immunoassay and western blotting were assembled using this HIV-2 antigen [F. Brunvezinet et al, Lancet, i, 128 (19
87)] Enzyme-linked immunosorbent assay immobilizes the HIV-2 antigen, reacts it with the test serum, removes unreacted serum components, and reacts with an enzyme-labeled antibody specific to the reacted human antibody. Let it. After the reaction, remove the unreacted enzyme-labeled antibody, add a substrate specific to the enzyme, and use HIV depending on the degree of color development.
This is a method for examining the presence or absence of an antibody against -2. Western blotting is a method in which the HIV-2 antigen is electrophoresed, transferred to a nitrocellulose membrane, reacted with a test serum, and the reacted antibody is colored in the same manner as in the enzyme immunoassay to be detected as a band. It is.

[Problems to be solved by the invention]

As described above, each method for detecting the HIV-2 antibody is devised in sensitivity and specificity, but still has practical problems. First, the reaction operation is complicated in three steps and four steps. Second, special equipment is required for reaction measurement. The above problems are important problems to be solved when measuring a large number of test sera.

[Means for Solving the Problems] The present invention provides a novel reagent for detecting an HIV-2 antibody which solves the problems found in the above-described method for detecting an HIV-2 antibody, and comprises the steps of: The method is characterized by using an indirect agglutination reaction. That is, the present invention provides a method for producing an HIV-2 antigen by treating HIV-2 obtained by culturing HIV-2 producing cells with an anionic surfactant, and carrier particles sensitized with the antigen. The present invention relates to an indirect agglutination reagent for detecting an HIV-2 antibody, which is mainly used for

HIV-2 antigen can be obtained according to the following method. That is, HIV-2 producing cells are cultured, and HIV-
After separation of HIV-2, HIV-2 antigen can be obtained by treating HIV-2 with an anionic surfactant.

The above HIV-2 producing cells include all cell lines producing HIV-2. For example, like HIV, HIV-2 is known to infect cells having the T4 antigen, which is the surface antigen of human T cells [F. Clovel et al, Science, 2
33, 343 (1986)].
By infecting the cells, HIV-2 producing cells can be obtained. In particular, the known cell lines HUT78 and CEM
It is known that when infected with HIV-2, these HIV-2-infected cells become HIV-2 producing cells that can be permanently subcultured, and these cell lines are used in the present invention. be able to.

[F. Clovel et al, Science, 233, 343 (1986)]. HI above
Culture of V-2 producing cells can be performed by a usual method.

As the medium, any of various nutrient media used for ordinary cell culture can be used. A preferred medium is RP
Examples thereof include a medium in which MI 1640 medium, Eagle's minimum essential medium (MEM medium), and the like are modified using serum replacement fluid such as fetal calf serum (FCS) and calf serum. The culture conditions are not particularly different from those used for ordinary cell culture, and generally a temperature of about 36 to 38 ° C and a pH of 6.4 to 7.6 can be employed. Culture is usually performed by exchanging the liquid every 3 to 5 days, whereby the desired cells can be satisfactorily grown.

In the present invention, as the HIV-2 used as a raw material of the HIV-2 antigen component, one isolated from the culture supernatant of the above-mentioned HIV-2 producing cell by a conventional method is used. HIV-2
Is performed by a usual centrifugation method or the like. Particularly, using an ultracentrifugal density gradient method or the like is advantageous because HIV-2 can be obtained with high purity.

The HIV-2 antigen can be obtained by treating the above HIV-2 with an anionic surfactant.

Examples of the anionic surfactant include sodium alkyl sulfate and sodium bile acid.

In the case of using alkyl sodium sulfate, those having 10 to 20 alkyl chains are preferably used. The use of sodium alkyl sulfate is preferred in that a solubilized antigen having a high aggregating titer can be obtained among the anionic surfactants.

As a method for obtaining the antigen, an anionic surfactant 0.01
Buffer containing ~ 3.0% is added to the precipitate of HIV-2, and about 25-10
Treat at a temperature of 0 ° C. for 3 to 60 minutes. The supernatant obtained by centrifuging this lysate is used as HIV-2 antigen.

Next, the indirect agglutination reagent for detecting the HIV-2 antibody of the present invention will be described.

The carrier particles in the present invention may be any carriers as long as they can be used for the indirect agglutination reaction, and include erythrocytes of animals such as humans, sheep and chickens, and gelatin particles (Japanese Patent Application Laid-Open No. 57-15 / 1982).
3658) can be exemplified. In particular, gelatin particles have excellent advantages as a carrier for an indirect agglutination reaction for detecting HIV-2 antibody, for example, having little nonspecific reaction and being able to add properties depending on the application.

The method of sensitizing the carrier particles with the antigen component of HIV-2 may be a known method for sensitizing general antigens. For example, a method using a so-called coupling agent such as tannic acid, glutaraldehyde, bisdiazobenzidine, tolylene diisocyanate, difluorodinitrobenzene, carbodiimides, quinones, and chromium chloride, or a method of performing physical adsorption, or the like. I can do it.

The thus obtained HIV-2 antigen-sensitized particles are usually freeze-dried by a conventional method, reconstituted, and combined with a test sample (usually serum) diluted solution, standard serum, unsensitized carrier, etc. to obtain HIV-sensitized particles.
It is used for detection and measurement of HIV-2 antibody as an indirect agglutination reagent for detecting -2 antibody.

The preparation of the kit may be carried out according to a usual method. For example, sensitized particles, unsensitized carrier, standard serum (positive serum for control)
Then, a kit can be prepared by combining a solution for diluting the test serum and, if necessary, a solution for dissolving the lyophilized product of the sensitized particles.

[Action]

An equal amount of a two-fold serial dilution of the HIV-2 antigen-sensitized particle suspension and the serum of an AIDS patient is added to a well of a 96-well microplate, mixed, and allowed to stand at room temperature. After 1 to 3 hours, when observing each well, HIV-2 antigen-sensitized particles spread all over the bottom in the hole with a low dilution factor, and an aggregation image was seen. No image is observed. HIV-
Aggregation images of particles not sensitized to 2 antigens are not observed in any of low to high dilution serum. In addition, when the same test is performed on healthy human serum, no aggregated image is seen in the HIV-2 antigen-sensitized particles and the unsensitized particles. This indicates that the antigen on the HIV-2 antigen-sensitized particles specifically reacts with the HIV-2 antibody present in the serum of the AIDS patient.

The effects of the present invention will be described with reference to examples described below.

[Experimental Example 1] Selection of surfactant Samples below were prepared.

Sample 0.5% in 5 × 10 ⁷ PFU HIV-2 virus pellet
Polyoxyethylene (10) octyl phenyl ether
3 ml was added, the mixture was stirred at 37 ° C. for 30 minutes, centrifuged at 10,000 × g for 30 minutes, and the supernatant was used as a sample.

Sample A sample was prepared in the same manner as described for the sample except that 0.5% polyoxyethylene (9) octyl phenyl ether was used instead of 0.5% polyoxyethylene (10) octyl phenyl ether.

Sample A sample was prepared in the same manner as in the sample except that 0.075% sodium n-dodecyl sulfate was used instead of 0.5% polyoxyethylene (10) octyl phenyl ether.

Sample A sample was prepared in the same manner as described for the sample except that 0.075% sodium n-tetradecyl sulfate was used instead of 0.5% polyoxyethylene (10) octyl phenyl ether.

Sample A sample was prepared in the same manner as described for the sample except that 0.5% sodium deoxycholate was used instead of 0.5% polyoxyethylene (10) octyl phenyl ether.

Method Using each sample, according to the method described in Production Examples, HIV
-2 antigen-sensitized gelatin particles were produced. Agglutination tests were performed using HIV-2 infected sera with antibodies to HIV-2.

Results The results are shown in Table 1.

In the agglutination reaction in the table, a negative indicates that no clear agglutination image was recognized, a positive indicates that an agglutination image was recognized, and the agglutination titer indicates the final dilution of serum observed from the agglutination image. Table 1 shows that the anionic surfactant solubilized the HIV-2 antigen suitable for the agglutination reaction. In particular, the antigen solubilized by the sodium alkyl sulfate shows a high aggregating titer, which proves to be suitable for the production of the HIV-2 antigen.

〔Example〕

Production Example 1 (1) Preparation of HIV-2 Antigen HIV-2 was infected to Molt-4 cells, and cells established as HIV-2 producing cells were replaced with RP containing 10% fetal bovine serum.
Inoculate MI-1640 medium and incubate in air containing 5% carbon dioxide.
C. for 4 days.

Twenty liters of this culture was centrifuged at 3000 rpm for 10 minutes to remove cells, and a supernatant was obtained. The supernatant was layered on a 0-50% sucrose density gradient previously prepared in a continuous zonal rotor, and ultracentrifuged at 30,000 rpm while circulating.

The obtained virus fraction was layered again on a continuous density gradient of sucrose of 20 to 55% prepared in a tube for centrifugation, and ultracentrifuged at 25,000 rpm for 4 hours by a swing rotor. The obtained virus fraction was centrifuged at 45000 rpm for 60 minutes to precipitate the virus, and a phosphate buffered saline solution containing 0.075% sodium n-dodecyl sulfate was added to the precipitate. Float the precipitate, stir at 37 ° C for 30 minutes to dissolve,
The supernatant obtained by centrifugation at 13000 rpm for 30 minutes was used for HIV-
Two antigens were used.

(2) Preparation of Sensitized Particles Gelatin particles produced by the method described in Example 1 of JP-A-58-113754 were prepared by mixing pH 6 containing 5 ppm of tannic acid.
0.15 M phosphate buffered saline (hereinafter 0.15 M PBS (pH 6.
This was dispersed in 0.15 M PBS (pH 7.2) so that the particle concentration became 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 centrifugation, and then dispersed in 0.15 M PBS (pH 6.0) to 5%. On the other hand, the HIV-2 antigen prepared in (1) was
Dilute 50-fold with PBS (pH 6.0), mix an equal volume of this diluted solution with the tannic acid-treated particle dispersion, and heat at 37 ° C for 40 minutes to sensitize the particles to HIV-2 antigen. I let it. The sensitized particles are washed three times with physiological saline, and then suspended and dispersed (1920 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)
g glycine 1.5 g, thimerosal 30.01 g and normal rabbit serum
A solution prepared by dissolving 1.0 ml in purified water so that the total volume becomes 100 ml)
Was dispersed. This dispersion was freeze-dried to obtain a freeze-dried product of HIV-2 antigen-sensitized gelatin particles.

(3) Preparation of test serum dilution solution Disodium hydrogen phosphate / 12 hydrate 1920 g, potassium dihydrogen phosphate 0.291 g, sodium chloride 0.438 g, gum arabic 0.25 g, sodium azide 0.15 g to make a total volume of 100 ml. Was dissolved in purified water to obtain a serum diluted solution.

(4) Preparation of kit In addition to lyophilized sensitized particles, lyophilized antigen-unsensitized particles and lyophilized control positive serum were separately prepared. The solutions were combined to form an HIV-2 antibody detection reagent kit.

Production Example 2 Sheep erythrocytes fixed with formalin according to a conventional method were 5V / V%
An equal volume of a solution of 0.001 W / V% tannic acid in 0.15 M PBS (pH 6.0) is added to the solution suspended in 0.15 M PBS (pH 6.0), and the mixture is occasionally stirred at 37 ° C. for 15 minutes. Reacted. After washing the blood cells with a physiological saline solution, the cells are washed with 0.15 M PBS (pH 6.0) at 5 V
/ V% to obtain a tannic acid-treated blood cell fluid.

0.15 M PBS added to HIV-2 antigen prepared in Production Example 1 (1)
(PH 6.0), the mixture was diluted 50-fold, an equal amount of this diluted solution was added to the tannic acid-treated blood cell solution, and the mixture was heated at 37 ° C. for 30 minutes. Blood cells were centrifuged, washed twice with 0.15 M PBS (7.2), suspended in a suspension dispersion, and lyophilized to obtain a freeze-dried sensitized blood cell.

1920 g of dinarium hydrogen phosphate / 12-hydrate, 0.291 g of potassium dihydrogen phosphate, 0.438 g of sodium chloride, gum arabic
0.25 g, sodium azide 0.15 g, normal rabbit serum 1.0 ml
It was dissolved in purified water so as to have a volume of 00 ml to obtain a serum dilution solution.

A freeze-dried product of sensitized blood cells, a dried product of unsensitized blood cells, a positive serum for control, and the above serum diluent were combined to prepare a reagent kit for detecting an HIV-2 antibody.

〔Example〕

Using the reagent kits obtained in each of the production examples, an indirect agglutination test was performed on various test sera. Table 2 shows the obtained results.

These results indicate that the indirect agglutination method has exactly the same detection sensitivity and specificity as the fluorescent antibody method in detecting the HIV-2 antibody.

〔The invention's effect〕

With the reagent of the present invention, a large number of samples can be rapidly measured by simple operations. For example, in the case of the fluorescent antibody method or the enzyme antibody method, washing and addition of several operations are required until the result is obtained from the start of the test, but when using the reagent of the present invention, it is only necessary to add the diluted serum to the diluted serum. What is necessary is just to stand still until the result comes out, and it is very simple. In addition, the result can be determined with the naked eye without using any special equipment. Moreover, the HIV-2 antigen used for the preparation of the reagent was treated with a surfactant,
It is highly safe because it has no infectivity. These points are suitable for testing a large number of specimens.

When the HIV-2 antibody is detected, it is considered that HIV-2 is retained in the body.
(2) Inspection of the presence or absence of the antibody is very clinically useful because infection due to contact and blood transfusion can be prevented.

Continuation of the front page (56) References JP-A-62-182662 (JP, A) JP-A-64-468 (JP, A) JP-A-1-160998 (JP, A) JP-A-1-163198 (JP, A) , A) Tokuyo Sho 63-502242 (JP, A) Tokuyo 1-503462 (JP, A) Tokuyo 1-502119 (JP, U) Lancet 1 [8525] (1987), P128-132

Claims (4)

(57) [Claims] 1. A method for producing a human immunodeficiency virus type 2 antigen, which comprises treating a human immunodeficiency virus type 2 cell obtained by culturing cells producing human immunodeficiency virus type 2 with an anionic surfactant. . 2. The method for producing a human immunodeficiency virus type 2 antigen according to claim 1, wherein the anionic surfactant is sodium alkyl sulfate. 3. A human immunodeficiency virus type 2 antigen obtained by culturing human immunodeficiency virus type 2 producing cells and treating the obtained human immunodeficiency virus type 2 with an anionic surfactant. An indirect agglutination reagent for detecting human immunodeficiency virus type 2 antibody, which is mainly composed of sensitized particles sensitized to particles. 4. The reagent according to claim 3, wherein the anionic surfactant is sodium alkyl sulfate.