JPS61200471A - Bovine leukemia virus antibody detection reagent - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) Bovine leukemia virus has been confirmed to be a causative virus that causes lymphoma, which is a type of leukemia. Therefore, it is important to detect the presence or absence of this virus infection in order to prevent the spread of infection.

The present invention utilizes a reaction in which carrier particles sensitized with bovine leukemia virus or its antigen components agglutinate with bovine leukemia virus antibodies contained in the serum of mammals infected with bovine leukemia virus, to determine the presence or absence of bovine leukemia virus infection. The present invention relates to a reagent for detecting bovine leukemia virus antibodies for diagnosis.

(Prior Art) As testing methods, agar precipitation reaction method, complement fixation reaction method, fluorescent antibody method, enzyme immunoassay method, radioimmunoassay method, etc. have been developed.

(Problems to be Solved by the Invention) These methods were complicated to operate, took time, and required special measuring equipment.

(Means for Solving the Problems) The present invention provides a method for easily and inexpensively measuring infection with bovine leukemia virus. The presence of antibodies to the bovine leukemia virus easily agglutinates bovine leukemia virus antibodies, and tests using this agglutination reaction are several steps higher in specificity and sensitivity than the conventionally widely used agar gel precipitation reaction method. This was done after discovering that it can rotate.

That is, the present invention relates to an indirect agglutination reaction reagent for detecting bovine leukemia virus antibodies, in which carrier particles are sensitized with bovine leukemia virus or its antigen components.

Bovine leukemia virus is a type of retrovirus that infects the lymphocytes of cattle, and is also called 9BLV.

The bovine leukemia virus can be obtained by any known method, such as Fatal Lamb Kidney.
You can culture established cell lines such as Cell (FLK), separate the cells from the culture solution, and isolate the bovine leukemia virus from these cells or the culture supernatant. You can receive a condominium from.

After culturing, to obtain the proliferated virus from the cells, the culture solution is collected by centrifugation, and the precipitate is diluted with Tris-HCl buffer.
Wash with NaC2-EDTA, phosphate buffered saline, etc., and treat with a surfactant such as Nonidet P2O, Triton X100, etc. to destroy cells and viruses. 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 convenient to separate the bovine leukemia virus fraction by ultracentrifugation density gradient method, since the bovine leukemia virus can be obtained with high purity. Isolated viruses are usually destroyed with surfactants such as Nonidede P40 and Triton X100 and used as virus antigens. however,
It can also be used as a virus antigen without destroying the virus by simply losing its replication function.

The carrier particles may be those for indirect agglutination reactions, such as excited red blood cells of humans, sheep, chickens, etc., microorganisms such as Serratia bacteria, gelatin particles (Japanese Patent Application Laid-Open No. 153658/1982), polystyrene latex, and kaolin. , charcoal powder, etc. can be used.

The method for sensitizing carrier particles with bovine leukemia virus or its antigenic component may be any known method for sensitizing general antigens. This can be carried out by a method using a so-called coupling agent such as Orodinitrobenzene, carbodiimides, quinones, and chromium chloride, or by a method of physical adsorption.

The 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 used for measurement. .

The method for measuring bovine leukemia virus antibodies using the reagent of the present invention may also be carried out by a conventional method using an indirect agglutination reaction. 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, mixing the test serum and sensitized particles on a slide plate,
It is also possible to observe the state of aggregation after minutes.

(Function) Since the carrier particles are sensitized with bovine leukemia virus or its antigen component, these particles aggregate in the presence of bovine leukemia virus antibodies.

Production Example 1 (1) During preparation of bovine leukemia virus antigen Fetallam, a leukemia virus producing cell line
b kidn@y cells were inoculated into Eagle's MEM medium containing 10% fetal bovine serum and cultured at 37°C for 4 days. This culture solution 20j was centrifuged at 13,000 rpm for 10 minutes to remove cells and obtain supernatant fluid.

This supernatant liquid was prepared in advance in a continuous zonal rotor.
~504 layered on the sucrose density gradient and circulated to 300
Ultracentrifugation was performed at 0 Q rpm.

The obtained virus fraction was layered again on a 15-50% continuous density gradient prepared in a centrifuge tube 1, and ultracentrifuged at 25,000 rpm for 4 hours using a swing rotor. Obtained virus fraction molecules 45,000
The virus was precipitated by centrifugation at rpm for 60 minutes, and phosphate buffered saline containing KO, 5% Nonidet P-40 was added to the precipitate. The precipitate was suspended, stirred in ice water for 30 minutes to dissolve, and centrifuged at 13,000 rpm for 30 minutes. The resulting supernatant was used as a bovine leukemia virus antigen.

(2) Preparation of sensitized particles Disodium hydrogen phosphate decahydrate 1.920.91,
Potassium dihydrogen phosphate 0.291g, sodium chloride 0
．． 438.9, gum arabic 0.25.9. Glycine 1
．． 5g, thimerosal 0.01.9 and normal rabbit serum 1
, Ornlf was dissolved in purified water to a total volume of 100 ml to obtain a suspended dispersion.

Disodium hydrogen phosphate 1.920.9° Potassium dihydrogen phosphate 0.291.9. Sodium chloride 0
．． 4381 gum arabic 0°25g, sodium anodide 0.151 normal rabbit serum 1. Ornl, sheep red blood cell membrane disrupted solution2. Ornl and bovine red blood cell membrane disrupted solution 1. Oru
A serum dilution solution was prepared by dissolving lf in purified water to a total volume of 100 ml.

Sheep red blood cells fixed with formalin according to a conventional method were mixed at 5 v/
0.15 M phosphate buffered saline (0.15 MPBS (7.2)) at pH 7.2 to
An equal volume of 0.001 W/V 4 tannic acid in 0.15 MPBS (7,2) solution was added to the suspended solution, and the mixture was reacted at 37° C. for 15 minutes with occasional stirring. After washing the blood cells with physiological saline, 0.15MPBS (7,2
) to obtain a tannic acid-treated blood cell solution.

0.15MP to the bovine leukemia virus antigen prepared in (1)
BS (7,2) was added to dilute it 100 times, and an equal amount of this diluted solution was added to the tannic acid-treated blood cell solution and incubated at 37°C.
The mixture was heated for 30 minutes. Blood cells were centrifuged to 0.15 M
It was washed twice with PBS (7,2), suspended in a floating dispersion, and freeze-dried.

This lyophilized product was combined with a dried product of naive blood cells, a positive serum for control, and a serum dilution solution to prepare a reagent kit for detecting live leukocyte virus antibodies.

Production example 2 Sodium tetraborate decahydrate 1.91. l lactic acid 0.8
5mA! , sodium chloride 0.45I, glycine 1.5
g, sucrose 4. OI and normal rabbit serum 1. 0ml was dissolved in purified water to a total volume of 100ml to prepare a dispersion suspension.

Sodium tetraborate decahydrate o, sg, potassium dihydrogen phosphate o, s, p, sodium chloride 0119° polyethylene glycol 60000.15 y, /
IJ vinylpyrrolidone -300,15,9, sodium azide 0.15, li', normal rabbit serum 0.3M and normal chicken serum 0,4. One total amount of rnlf and 10 total amounts were added and dissolved, and this was used as a serum dilution solution.

Fixed chicken red blood cells were prepared by treating chicken red blood cells with cyformin using a conventional method, and the blood cells were adjusted to pH 7, 0.
1 M phosphate buffer (0.1 M PH (7.2
)) at 5 v/v 4. Tolylene diisocyanate (TDI) emulsion solution was added to this blood cell suspension at a final concentration of 0. The mixture was added to the solution at a concentration of 0.5 to 4.0 and stirred at 37° C. for 40 minutes. Blood cells at 0.05 MPB (
6.4) to obtain a TDI-treated hemocyte precipitate.

The bovine leukemia virus antigen prepared in (1) of Production Example 1 was diluted 100 times with 0.3 M PH (8.5), and this diluted solution was added to the above TDI-treated hemocyte sediment so that the hemocyte concentration was 5 v/v. Add to 4 and mix well.
The reaction was carried out by heating at 37° C. for 45 minutes. Blood cells were centrifuged, washed once with physiological saline and once with the dispersion suspension, suspended in the suspension suspension, and freeze-dried.

This is a reagent kit for detecting antibodies.

Production Example 3 Gelatin particles produced by the method described in Example 1 of JP-A-58-113754 were placed in 0.15 MPBS (7.2) containing 5 ppm of tannic acid at a particle concentration of 2.5. The mixture was dispersed evenly and heated at 37°C for 10 minutes. The particles were collected by centrifugation, washed with physiological saline by centrifugation, and then washed with 0.15 MPBS (
7.2) were dispersed into 5 pieces.

On the other hand, the bovine leukemia virus antigen prepared in (1) of Production Example 1 was added to 0. 1 at 15M PBS (7.2)
An equal volume of this diluted solution was mixed with the tannic acid-treated particle dispersion and heated at 37° C. for 40 minutes to sensitize the particles to the bovine leukemia virus antigen. The sensitized particles were centrifugally washed with physiological saline and then dispersed in a suspension suspension prepared in the same manner as in Production Example 1 (2). This dispersion was freeze-dried to obtain a freeze-dried product of gelatin particles sensitized with bovine leukemia virus antigen.

Since the carrier was gelatin particles, the procedure of Production Example 1 (2
) A serum dilution solution was prepared in the same manner as (Effects of the Invention) By using the reagent of the present invention, bovine leukemia virus antibodies could be detected with much higher sensitivity and higher sensitivity than the conventional agar cell precipitation reaction method. Can be measured with precision. The measurement method using the reagent of the present invention has the advantage that it is simple and can be easily carried out anywhere since it does not require special equipment.

Patent applicant: Fujirebio Co., Ltd. Representative: Masahiro Tanaka, patent attorney

Claims (1)

[Claims] An indirect agglutination reaction reagent for detecting bovine leukemia virus antibodies, in which carrier particles are sensitized with bovine leukemia virus or its antigen components.