JP3033774B2 - Indirect agglutination reagent for detection of adult T-cell leukemia virus antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a component obtained by treating adult T-cell leukemia virus (hereinafter referred to as HTLV-I) with a surfactant and HTLV-I.
The present invention relates to an indirect agglutination reagent for detecting an HTLV-I antibody using carrier particles sensitized with an env gp-21 protein.

[Conventional technology]

HTLV-I is known as a causative virus that causes T-cell lymphoma, a type of leukemia, and testing for the presence of this virus is important in clinical, epidemiological and transfusion situations.

As a method for testing for the presence or absence of HTLV-I infection, an indirect agglutination immunoassay, a fluorescent antibody method, an enzyme immunoassay, a radioimmunoassay, and the like have been developed. Is easy and inexpensive to measure. As an indirect agglutination reagent used for the measurement of HTLV-I, a test reagent using carrier particles treated with HTLV-I with a surfactant and sensitizing the resulting component (JP-A-60-1985)
44870) is already known.

[Problems to be solved by the invention]

However. In the case of a conventional reagent, a prozone phenomenon in which aggregation does not occur even in the case of a positive sample sometimes occurs when the sample is in excess of an antibody during measurement. for that reason,
The problem of a false negative in which even a positive sample is determined to be negative has occurred.

[Means for solving the problem]

The present inventors have studied to overcome the conventional disadvantages, and as a result, have found that one of the proteins constituting HTLV-I, env gp-21,
The present inventors have found that an indirect agglutination reagent for detecting HTLV-I antibodies using carrier particles sensitized with a component obtained by treating a protein and HTLV-I with a surfactant can suppress the prozone phenomenon, and completed the present invention. .

The present invention is an indirect agglutination reagent for detecting HTLV-I antibody using a component obtained by treating HTLV-I with a surfactant and carrier particles sensitized with env gp-21 protein constituting HTLV-I.

In producing a component obtained by treating HTLV-I with a surfactant, it is necessary to first obtain an HTLV-I antigen.To obtain the HTLV-I antigen, a cell line is cultured, It can be obtained by separating cells from the culture solution and separating from the cells, or by separating HTLV-I from the culture supernatant. As the cells to be transformed, for example, MT-2,
HUT-102 or the like can be used.

In order to separate the cells from the culture solution and separate them from the cells, the precipitate obtained by centrifuging the culture solution after culturing is washed with Tris-HCl buffer-NaCl-EDTA, a phosphate buffered saline solution, and the like. The cells and the virus are destroyed by treating with a surfactant, and the supernatant obtained by removing the precipitate of the disrupted cells and the virus by centrifugation can be used as a virus antigen.

When obtaining a virus from a culture supernatant, if the HTLV-I fraction is separated by ultracentrifugation density gradient method, HTLV
-I alone, and according to this method HTLV
-I can be obtained with high purity. The isolated virus is usually destroyed with a surfactant and used as an antigen. However, the virus can be used as an antigen without destroying the virus itself only by losing the growth function of the virus.

The env gp-21 protein constituting HTLV-I can be obtained by treating the HTLV-I with a surfactant and purifying a component obtained by lectin chromatography.

Surfactants that can be used in obtaining the antigen include Nonidet P40 and Triton # 100.

Components obtained by treating HTLV-I with a surfactant and en
v The method of sensitizing the carrier particles to the gp-21 protein can be a method by forming a chemical bond or a method by physical adsorption, such as tannic acid, glutaraldehyde, bisdiazobenzidine, Range isocyanate, difluorodinitrobenzene,
It can be achieved by a method using a so-called coupling agent such as carbodiimides, quinones, and chromium chloride.

In preparing the reagent, a predetermined amount of the component obtained by treating HTLV-I with a surfactant and env gp-21 protein are mixed, and sensitization can be performed simultaneously using the above method. First, one of the components obtained by treating HTLV-I with a surfactant or the env gp-21 protein is sensitized, and then the other is sensitized to separately sensitize the components. it can. Furthermore, the reagent of the present invention can also be prepared by preparing carrier particles comprising only the respective antigen components and mixing both carrier particles.

Examples of the carrier particles include erythrocytes of animals such as humans, sheep and chickens, microbial cells such as Serratia, gelatin particles (JP-A-57-153658), polystyrene latex, kaolin, charcoal powder, and the like. The particles used can be used.

Example 1 HUT-102 cells, which are HTLV-I producing cell lines, were inoculated into RPMI-1640 medium containing 10% fetal bovine serum, and cultured at 37 ° C. for 4 days in air containing 5% carbon dioxide. . This culture was centrifuged at 3000 rpm for 10 minutes to remove cells, and a supernatant was obtained. This supernatant was layered on a 20-55% sucrose gradient previously prepared in a continuous zonal rotor, and ultracentrifuged at 30,000 rpm while circulating. The obtained virus fraction was again layered on a continuous density gradient of 20 to 55% prepared in a centrifuge tube, and ultracentrifuged at 25,000 rpm for 4 hours by a swing rotor. 45 of the virus fraction obtained
The virus was precipitated by centrifugation at 000 rpm for 60 minutes, and 0.5% Nonidet-P40-containing phosphate buffered saline was added to the precipitate. The precipitate is suspended, dissolved in ice water by stirring for 30 minutes, and the supernatant obtained by centrifugation at 13000 rpm for 30 minutes is subjected to HTLV-
I antigen.

The prepared HTLV-I antigen (350 ml, 400 μg / ml) was dialyzed against 10 ml Tris-HCl buffer (pH 8.3), and subjected to high-speed centrifugation at 10,000 rpm for 10 minutes. The column was packed in a lentil-lectin affinity column equilibrated in .3), washed with the above buffer solution, eluted with methyl αD mannoside, and a portion exhibiting an absorbance of 280 nm was collected. Further, the obtained fraction was dialyzed against 10 mM Tris-HCl buffer (pH 8.3) to obtain 9 mg of purified env gp-21 protein.

Example 2 Gelatin particles were dispersed in phosphate-buffered saline (pH: 7.2) containing 5 ppm of tannic acid so as to have a particle concentration of 2.5%, and then dispersed at 37 ° C. for 15 minutes. Heated. The particles were then centrifuged and washed thoroughly with saline 0. The obtained particles were dispersed in PBS at a concentration of 5%, and this dispersion was mixed with the env gp-21 protein obtained in Example 1, and the mixture was heated at 37 ° C. for 40 minutes to give sensitized gelatin. Particles were prepared. In a similar manner, gelatin particles sensitized only with the HTLV-I antigen were prepared. Each of the prepared sensitized particles was mixed at a ratio of 1: 1 to obtain a reagent for detecting HTLV-I antibody.

Example 3 env gp-21 protein obtained by the method of Example 1 and
The HTLV-I antigen was mixed at a ratio of 5: 1, and the mixture was sensitized to gelatin particles in the same manner as in Example 2 to obtain a reagent for detecting an HTLV-I antibody.

Comparative Example 1 Using the reagent prepared in Example 2 and a reagent prepared by a conventional method, an indirect agglutination test was performed on a false-negative specimen of an HTLV-I antibody. The results obtained are shown in the table below.

Comparative Example 2 Using the particles prepared in Example 2 and the particles prepared by the conventional method, an indirect agglutination test was performed on the antibody-rich HTLV-I antibody.

 The results obtained are shown in the table below.

[Effects of the Invention] The indirect agglutination reagent of the present invention is properly determined as positive even in a test of an HTLV-I antibody, even if a conventional indirect agglutination reagent may be determined to be negative due to the prozone phenomenon. In addition, it is characterized by high measurement sensitivity. Therefore, according to the present invention, the inspection system of the HTLV-I antibody inspection can be significantly improved.

Continuation of front page (56) References JP-A-60-44870 (JP, A) JP-A-59-35143 (JP, A) JP-A-63-277970 (JP, A) JP-A-2-1559 (JP) , A) J. Virol. , 63 (11) (1989) pp. 4952-4957. Immunol. , 142 (3) (1989) p971-978.

Claims (2)

(57) [Claims] 1. A component obtained by treating an adult T-cell leukemia virus with a surfactant, and a component obtained by treating the adult T-cell leukemia virus.
v Indirect agglutination reagent for detecting adult T-cell leukemia virus antibody using carrier particles sensitized with gp-21 protein. 2. The reagent according to claim 1, wherein the carrier particles are gelatin.