JPH0460458A - Indirect agglutination reaction reagent for adult T-cell leukemia virus antibody detection - Google Patents

Abstract

PURPOSE:To enable improvement of the precision in inspection by using a constituent obtained by subjecting an adult T cell leukosis virus to a surfactant treatment and a carrier particle of gelatin or the like obtained by sensitizing env gp-21 protein of the same virus. CONSTITUTION:Env gp-21 protein constituting an adult T cell leukosis virus HTLV-I is obtained by refining a HTLV-I antigen constituent by lectin chromatography. An HTLV-I antigen sensitizing particle is subjected to freeze drying and provided for measurement in combination with a renaturation liquid, a liquid for serum dilution, a standard serum, an unsensitized carrier, a nonspecific reaction absorbing liquid, etc. By this method, the precision in inspection can be improved.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to adult T-cell leukemia virus (hereinafter referred to as HTLV-I).
Components obtained by treating ) with a surfactant and HTL
The present invention relates to an indirect agglutination reaction reagent for detecting HTLV-I antibodies using carrier particles sensitized with env gp-21 protein, which constitutes VI.

[Conventional technology]

HTLV-t is a virus that causes T-cell lymphoma, a type of leukemia. Therefore, testing for the presence or absence of this virus infection is important clinically, epidemiologically, and during blood transfusion.

As testing methods, fluorescent antibody methods, enzyme immunoassays, radioimmunoassays, and the like have been developed. Further) ITLV-
H as a simple and inexpensive method to measure the presence or absence of infection.
A test reagent that uses carrier particles that have been treated with TLV-1 and sensitized with the resulting components, and utilizes the fact that these particles cause an agglutination reaction in the presence of HTLV-1 antibodies.
0-44870) is already commercially available.

[Problem that the invention seeks to solve]

However, with conventional reagents, the occurrence of the prozone phenomenon was rarely observed in areas where the dilution ratio of the specimen was low, and as a result, false negative results were sometimes obtained for specimens that were positive by other testing methods, such as the IF method.

[Means for solving problems]

As a result of studies to overcome the conventional drawbacks, the inventors of the present invention found that
enV g, one of the proteins that constitute HTLV-1
I(T) using carrier particles sensitized with components obtained by treating P-21 protein and HTLV-1 with a surfactant.
The present invention was completed by preparing an indirect agglutination reaction reagent for detecting LV-1 antibody and using it to detect the antibody, and confirming that extremely accurate measurement could be performed.

The present invention is an indirect agglutination reaction reagent for detecting HTLV-1 antibody, which uses a component obtained by treating HTLV-1 with a surfactant and carrier particles sensitized with env gp-21 protein, which constitutes HTLV-1.

In producing a component obtained by treating HTLV-7 with a surfactant, the first step is to obtain the HTLV-I antigen. HTLV-1 can be obtained by any known method, for example, by culturing established cell lines such as MT-2 and HUT-102, separating the cells from the culture medium, and extracting HTLV-1 from the cells or the culture supernatant. Just separate it. The HUT 102 is, for example, an NCI (Natio
nal Cancer Institute, Na
tional In5titude of Heal
Th.

Bethesda, Maryland, U, S, ^)
You can receive a condominium from. After culturing, to obtain the proliferated virus from the cells, centrifuge the culture solution and collect the precipitate in Tris-HCl buffer-NaCl-EDTA.
, wash with phosphate buffered saline solution, etc., and remove
Destroy cells and viruses by treating with a surfactant such as 2O, Triton X 100, etc. Then, the precipitate is removed by centrifugation, and the supernatant can be used as a virus antigen. On the other hand, when obtaining the virus from the culture supernatant, it is possible to isolate the HTLV-1 fraction by ultracentrifugation density gradient method.
This is convenient because LV-I can be obtained with high purity. The isolated virus is usually Nonide 1-P40. ) Destroy with a surfactant such as Rydon X100 and use as a virus antigen. However, it can also be used as a virus antigen without destroying the virus by simply losing its replication function.

Env gp-21 protein, which constitutes HTLV-1, can be obtained by purifying the HTLV-1 antigen component obtained by the above method using lectin chromatography.

The method for sensitizing carrier particles with HTLV-I or its antigen component and env gp-21 protein may be any known method for sensitizing general antigens, such as tannic acid, geltaraldehyde, bisdiazobenzidine, This can be carried out by a method using a so-called coupling agent such as tolylene diisocyanate, difluorodinitrobenzene, carbodiimides, quinones, and chromium chloride, or by a method of physical adsorption.

Also, HTLV-I or its antigenic component and env gp-21
When sensitizing protein to carrier particles, HTLV-I or its antigen component and env gp~21 protein can be mixed in a predetermined amount using the above method, and sensitization can be carried out simultaneously.
After sensitizing HTLV-I or antigen components, env gp
-21 protein can also be sensitized.

The carrier particles may be those for indirect agglutination reactions, such as red blood cells of animals such as humans, sheep, and chickens, microbial cells such as Serratia bacteria, gelatin particles (Japanese Patent Application Laid-Open No. 57-153658), polystyrene latex, kaolin, Charcoal powder etc. can be used.

The HTLV-1 antigen-sensitized particles thus obtained are usually freeze-dried using a conventional method, and then combined with a restoring solution, serum dilution solution, standard serum, unsensitized carrier, non-specific reaction absorption solution, etc., and subjected to measurement. Ru.

The method for measuring HTLV-I antibodies using the reagent of the present invention may also be a conventional measurement method using indirect agglutination reaction. HTLV-1 antigen-sensitized particles are added to each well, stirred, and allowed to stand for about 1 to 2 hours to observe the aggregation image of the sensitized particles. In addition to this method,
For example, it is also possible to mix the test serum and sensitized particles on a slide plate and observe the state of agglutination after 1 to 2 minutes.

Example 1 HTLV prepared by the method of JP-A-60-44870
-I antigen 350sl (400μg/ml) in 10ml)
IJ susu-acid buffer (pH 8,3) T: Dialysis 100
The supernatant obtained by high-speed centrifugation at 00 rpm TelO for 1 minute was loaded onto a lentil lectin affinity column equilibrated with Tris-HCl buffer (pH 8.3), washed with the buffer, and methyl αD mannoside was added. Elute with
A portion exhibiting an absorbance of 280 na+ was fractionated. Furthermore, the obtained dissolved fraction was added to a 10% tris-hydrochloric acid buffer (pH
8,3) to obtain 9 mg of purified env gp-21 protein.

Example 2 Gelatin particles sensitized with the env gp-21 protein obtained in Example 1 were prepared by the method of JP-A-59-35143. Also, gelatin particles sensitized only to the HTLVI antigen were prepared by the method described above.

Thereafter, the former and latter sensitized particles were mixed at a ratio of 1:1 and used as a reagent for detecting an HTLV-I antibody.

Example 3 The env gp-21 protein and HTLV-I antibody obtained by the method of Example 1 were mixed at a ratio of 5=1, and the mixture was sensitized to gelatin particles by the method of JP-A-59-35143. , was used as a reagent for detecting HTLV-1 antibodies.

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

Comparative Example 2 Using the particles prepared in Example 2 and particles prepared by a conventional method, an indirect agglutination test was conducted on antigen-excess HTLV-1 antibody.

The results obtained are shown in the table below.

[Effects of the Invention] The particles of the present invention showed positive results in indirect agglutination tests for HTLV-1 antibodies for samples that were falsely negative in the conventional method, and also showed positive results for samples with excess antigen. This contributes to improving the accuracy of the HTLV-■ antibody test.

Patent applicant: Fujirebio Co., Ltd.

Claims (2)

[Claims] (1) Components obtained by treating adult T-cell leukemia virus with a surfactant and envg of adult T-cell leukemia virus
An indirect agglutination reaction reagent for detecting adult T-cell leukemia virus antibodies using carrier particles sensitized with p-21 protein. (2) The reagent according to claim (1), wherein the carrier particles are gelatin particles.