JPH02181655A - Method for measuring anti-aids virus antibody and its measuring reagent kit - Google Patents

Abstract

PURPOSE:To rapidly and exactly measure the anti-AIDS virus antibody by bringing a carrier for agglutination and an AIDS virus antigen into reaction by using the anti- AIDS virus antibody measuring reagent for agglutination reaction, then adding a specimen thereto and further bringing the antibody to be measured into reaction. CONSTITUTION:The reagent kit consists of the sensitized carrier I formed by conjugating the AIDS virus antigen with the carrier for agglutination sensitized with the AIDS virus monoclonal antibody, a positive control II, a negative control III, and a buffer soln. IV for measurement. The liquid IV is put by 25mul each into the respective holes of a U plate and the liquid to be detected is put by 25mul each into the holes of the row A. The dilution times of the row J are than 1024 if the liquid mixtures are transferred by 25mul each to the row B. The similar dilution system is prepd. by using the liquids II and III in place of the liquid to be tested and the suspension of 5ml liquid IV per 1 vial of the carrier I is added by 25mul each to all the holes and after the liquid in incubated for 2 hours at room temp, the liquid is compared with the III. The quantity of the anti-AIDS virus antibody is determined by the max. dilution times at which the agglutinated image is admitted.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Application) The present invention relates to a method for measuring anti-AIDS virus antibodies using an agglutination reaction and a reagent kit for the measurement.

(Prior Art) AIDS virus antigen refers to an antigen derived from the AIDS virus, and anti-AIDS virus antibody refers to an antibody that reacts with the AIDS virus antigen or an antibody that reacts with the AIDS virus itself.

The AIDS virus is HIV (Human Immun
ode Ficiency Virus), HTLV
(Old +man T-cell Lympho
Tropic Virus).

LAV (Lymphadenopathy As5o
Also called mediated virus. According to the present invention, this includes AIDS-related viruses.

Antibody tests, antigen tests,
There is a method of detecting AIDS virus genes using DNA probes.

The principle of the antibody test that can determine the presence or absence of AIDS virus infection is that anti-AIDS virus antibodies produced in the blood of carriers due to infection are detected using AIDS virus antigens prepared in advance, and enzyme immunoassay (ELISA) is used. )
and agglutination methods have been developed and are used for infectious screening tests such as donated blood and clinical tests.

[Problem that the invention seeks to solve]

The present inventors carried out the hemagglutination reaction method using red blood cells sensitized with AIDS virus antigens according to conventional methods.
Attempts were made to measure anti-AIDS virus antibodies in specimens, but sufficient sensitivity was not obtained and the results were not necessarily satisfactory (the cause is unknown).

As a result of various studies, we decided to use a method in which red blood cells sensitized with an anti-AIDS virus monoclonal antibody are reacted with an AIDS virus antigen, and then a sample is added to further react with the anti-AIDS antibody to be measured in the sample. The inventors have discovered that anti-AIDS virus antibodies can be measured quickly and accurately by this method, and have completed the present invention.

[Means for solving problems]

Since it is convenient for the measurement reagents according to the present invention to be prepared in the form of kits, the present invention will be described below using the kits as examples.

When the reagent of the present invention is made into a kit, it is composed of the following reagents. That is, (i) a sensitized carrier obtained by sensitizing an anti-AIDS virus monoclonal antibody to an agglutination carrier and then binding an AIDS virus antigen through an antigen-antibody reaction (
(hereinafter referred to as sensitized carrier) (ii) Standard anti-AIDS virus antibody positive control (hereinafter referred to as positive control) (1it) Negative control, (iv
) The kit is composed of a combination of measurement buffers.

(i) Sensitized carrier The sensitized carrier is one in which an anti-AIDS virus monoclonal antibody is sensitized to an agglutination carrier, and then an AIDS virus antigen is bound thereto by an antigen-antibody reaction.

The anti-AIDS virus monoclonal antibody used to sensitize the agglutination carrier is an antibody against the AIDS virus antigen or an antibody against the AIDS virus itself, which will be described in the next section, and must satisfy the following conditions.

(1) It recognizes a different type of antigen-determining site from that which can be recognized by the anti-AIDS virus antibody to be measured. (2) There is only one antigen-determining site on the antigen (there is only one antigen-determining site on the antigen). (Recognizing only one antigen-determining site) If this condition is met, no agglutination reaction will occur between the sensitized carriers.

Such anti-AIDS virus monoclonal antibodies include:
It is prepared by immunizing an animal with a highly purified AIDS virus antigen for immunization, collecting antibody-producing cells that produce antibodies against the AIDS virus antigen, and treating them with cell fusion and transformation methods.

Any known immunization method may be used. For example, a mixed emulsion of a highly purified immunogen and Freund's complete adipant is prepared, and the animal is injected subcutaneously or intramuscularly two to three times, and antibody-producing cells are collected several weeks later. The ability to produce antibodies against AIDS virus antigens differs depending on the animal used for immunization, and it is economically advantageous to select highly susceptible animal groups, such as guinea pigs, rabbits, sheep, chickens, and horses. As a method for recovering antibody-producing cells, Ficoll-Conray specific gravity centrifugation (J, Cl) is used to separate and recover B-lymphocytes from collected blood.
1n, Lab, Invest, 21.5uppl
.

77-89 (1968)) etc. are used.

The cell fusion method is performed by known means. That is, a fusion hybrid is formed between an antibody-producing cell and a myeloma cell, and the hybrid is cloned to produce an antibody specific to the cancer cell (i.e., a specific antigen having the above characteristics). Manufactured by selecting.

Antibody-producing cells include tile cells, lymph node cells, B-'
One example is the J-npa ball.

As myeloma cells, those derived from mice, rats, humans, etc. are used, for example. For example, Mousemi x o-
? P3U1, X63-Ag8-6.5.3, etc. are mentioned. Preferably, the antibody-producing cells and myeloma cells are derived from the same species of animal.

Cell fusion can be achieved, for example, by Gee, Galfer (G.

Ga1fre) C naci + - (Nature)
266, 550 (1977) or a method analogous thereto, specifically, the treatment is carried out in the presence of polyethylene glycol.

The transformation method is also carried out by known means.

For transformation, lymphotropic viruses such as Epst
Ein Barr virus (EB virus) is used. The virus transforms normal cells into proliferative cells (Nature, 269.420-422
(1977) ) known as a virus. Transformation is performed, for example, as follows. 895-8 Origin E
B virus (mycoplasma)! (obtained as the supernatant from the I-895-8 medium) is used to prepare an amount of virus capable of cell transformation in advance. An appropriate amount of the corrected virus is dropped into the culture medium of the recovered antibody-producing cells and brought into contact with the culture medium.

Examples of carriers for agglutination include red blood cells, latex, gelatin, and the like.

We will specifically explain the case in which red blood cells are used.

It is not necessary to select a particular animal as the red blood cell to sensitize with the anti-AIDS virus monoclonal antibody, but in order to produce a stable and highly sensitive reagent, type 0 red blood cells such as sheep, chicken (chicken) IJ, and human red blood cells are preferable.

After thoroughly washing the red blood cells with physiological saline, they are stabilized by treatment with geltal aldehyde, formalin, etc. Such red blood cells preferably have a diameter of about 5 to 15 μm.

It is known that fil anti-AIDS virus monoclonal antibodies can sensitize these red blood cells [Medical History, 78.7
59 (1970)). For this purpose, it is best to sensitize red blood cells and anti-AIDS virus monoclonal antibodies in a buffer solution using a tannic acid treatment method, etc., and this is generally done by mixing a red blood cell suspension and a solution containing anti-AIDS virus monoclonal antibodies.

This operation is carried out at a pH of 2.5 to 8.5, a temperature of 15 to 60°C, and a concentration of the antibody to be sensitized (i.e., protein concentration) of 280 nm.
It is preferable to use a concentration at which the absorbance at a wavelength of 0.91 to 0.2 is obtained. The sensitized red blood cells are preferably lyophilized and sealed, for example in a vial, preferably with the addition of a preservative such as sodium azide.

The AIDS virus antigen is bound to this anti-AIDS virus monoclonal antibody-sensitized red blood cell.

The method for preparing the AIDS virus antigen is as follows.

Transfer the AIDS virus (AIDS-related virus) to appropriate cells (
Cε14 cells, Molt-4 cells, etc.) and then cultured in an appropriate medium for several weeks. This culture solution is centrifuged to remove cells and collect the supernatant. Furthermore, after performing ultracentrifugation using a sucrose density gradient or the like to collect a virus fraction, the AIDS virus antigen can be extracted using a surfactant and collected as a supernatant.

As for the method of binding the antigen, 1 to 50 ng/- of AIDS virus antigen solution is added to 1 volume of 1 to 10% anti-AIDS virus monoclonal antibody-sensitized red blood cell solution, p.
Examples include a method in which the reaction is carried out at t16 to 8.10 to 37°C for about 1 to 5 hours.

(ii) Positive control The positive control for the present invention is for confirming that the measurement is being performed normally, and usually consists of a liquid containing anti-AIDS virus antibodies. Such anti-AIDS virus antibodies include anti-AIDS virus antibody-positive human serum that has been subjected to virus inactivation treatment, or
It is produced by immunizing an animal with a highly purified AIDS virus antigen and using the resulting antiserum.

The antiserum may be produced by a known method; for example, a mixed emulsion of highly purified AIDS virus antigen and complete Freund's adjuvant is prepared, injected into the animal's skin 5 to 6 times, and blood collected several weeks later. After coagulating at room temperature, the antiserum is obtained by standing overnight at 4°C and centrifuging at 3000 rpm for 20 minutes.

Since the ability to produce antibodies against AIDS virus antigens differs depending on the animal used for immunization, it is economical and convenient to select highly susceptible animal groups, such as guinea pigs, rabbits, sheep, chickens, and horses.

The positive control is sealed, for example, in a vial, preferably with the addition of a preservative such as sodium azide.

(iii) Negative control The negative control consists of a liquid that is negative for both anti-AIDS virus antibodies and AIDS virus antigens (e.g., human serum, physiological saline, etc.), for example, in a vial, preferably with a preservative such as sodium azide. It is preferable to add it and encapsulate it.

(iv) Measurement 1 Equilibrium The measurement buffer is used to dilute the sensitized carrier, positive control, negative control, and specimen, and is made of, for example, a phosphate buffer to prevent non-specific reactions. To prevent this from occurring, for example, stroma, animal serum, etc. may be added. It is also preferable to add a preservative such as sodium azide.

The outline of the measurement method is as follows.

After serially diluting the sample containing anti-AIDS virus antibodies, (i) the sensitized carrier solution (0.1-188%
Add and mix equal amounts of p) 16 to 8 under the conditions of p) 1.
0 to 30°C for about 1 to 5 hours (aggregation) Measure the agglutination image generated after the reaction.

(Effects) According to the method of the present invention, anti-AIDS virus antibodies in a sample can be measured more accurately and quickly than conventional methods.

Therefore, the method and reagent of the present invention are considered to be extremely useful in clinical tests or as research reagents.

(Examples) In order to explain the present invention in more detail, Examples and Experimental Examples are given, but the present invention is not limited by these in any way.

Reference Example 1 C8M cells were infected with an AIDS-related virus, brought into contact with RPM I-1640 medium containing 10% fetal bovine serum, and cultured at 37°C for 14 days in air containing 5% carbon dioxide. This culture solution was centrifuged at 300 rpm for 10 minutes to remove cells and obtain a supernatant. This supernatant was layered on a sucrose density gradient prepared in advance in a continuous zonal rotor, and centrifuged at 30,000 rpm.

The obtained virus fraction was centrifuged at 45,000 rpm to precipitate the virus, the precipitate was dissolved by adding phosphate buffered saline containing surfactant, and insoluble matter was removed by centrifugation. The serum was used as an AIDS-related virus antigen.

Example 1 After thoroughly washing sheep red blood cells with phosphate buffer, geltaraldehyde was added to a final concentration of 0.5%, treated overnight at 4°C, washed with the above buffer, geltaraldehyde was removed, and the cells were fixed. Red blood cells were obtained.

5% 18 A suspension of fixed red blood cells and an equal volume of tannic acid solution 5
~30mg/dJ? was added, stirred, and left at 37° C. for 15 minutes, followed by washing with physiological saline to obtain tannic acid-treated blood cells.

A purified anti-AIDS virus monoclonal antibody solution (A280 nm
(approximately 0.01) and left at pH 7.2 for 1 hour at 37°C to obtain anti-AIDS virus monoclonal antibody-sensitized blood cells. Then, it was thoroughly washed with phosphate buffer.

Anti-AIDS virus monoclonal antibody sensitized blood cells 1%9
8. Add an equal volume of AIDS virus antigen solution (1
~50ng/d) was mixed and left at room temperature for 3 hours.

Then, it was thoroughly washed with phosphate buffer.

The sensitized blood cells were dispensed and freeze-dried to obtain a reagent.

After freeze-drying, the sensitized red blood cells are chlorinated with animal serum (IJum-containing isotonic phosphate buffer (pH 7,2))
The solution was adjusted to 0.5% with a phosphate buffer containing 2% or more of sheep red blood cell stroma, and the agglutination reaction against anti-AIDS virus antibodies was measured using a microplate method.

Example 2 Kit composition The following four types of reagents are included in the anti-AIDS virus antibody measurement kit.

■ Sensitized sheep red blood cells: The contents of one vial consist of dried sensitized sheep red blood cells and a preservative of 0.5% (w8) when suspended in ■5 ml of phosphate buffered saline (PBS). ) A red blood cell suspension is obtained. 1.5 kg of sodium azide is added as a preservative.

■ Anti-AIDS virus antibody positive control; 1 vial contains 1 ml of sterile-filtered anti-AIDS virus antibody. The amount of anti-AIDS virus antibody of this solution determined by PHA method is 1:64 or more. 1 mg (0.1%) of sodium azide is added as a preservative.

■ Negative control 1 vial contains sterile-filtered human (male) serum or physiological saline that is negative for both anti-AIDS virus antibodies and AIDS virus antigens.

1 mg (0.1%) of sodium azide is added as a preservative.

(2) Phosphate buffer: One vial contains sterile-filtered isotonic phosphate buffer, pH 7.2 (PBS) 50-.

PBS contains solubilized stroma and animal serum to prevent non-specific reactions during testing.

Composition (5'Omg) Medium disodium phosphate (anhydrous) 395mg phosphoric acid -
Potassium 155mg Sodium chloride
255mg stroma
3% animal serum 1% sodium azide 50 mg pH 7.2 A 10-portion of the phosphate buffer is used for turbidity of sheep red blood cells sensitized with anti-AIDS virus monoclonal antibody, and the rest is used for diluting the test solution. Sodium azide 1 as a preservative
It is preferable to add mg (0.1%).

Example 3 A quantitative test was conducted using the reagents shown in Example 2 as follows.

■ Suspend sensitized sheep red blood cells in 5 vials of phosphate buffer.

■ Add 25μβ of phosphate buffer to each person on the U-plate using a dropper.

■ Using the dropper, test wave valley 25 is placed in the hole in row A.
Add μβ each. Transfer the mixed solution in column A to column B using a diluter, then move it from column B to column 0, and transfer it one after another until 3.
Do this until you reach the column. As a result, the test liquid is serially diluted 2 times, and the dilution factor in the 3 rows is 1.024 times.

■ Separately, create a dilution series for the positive control instead of the test solution.

(2) Using a dropper, add 25 μβ of a suspension of sensitized sheep red blood cells in phosphate buffer to all the holes.

■ Shake the plate with a micromixer for about 10 seconds, then incubate at room temperature for 2 hours.

■ Observation of controls: For S-type controls, agglutination is usually observed at 64-fold dilution or higher. Negative controls show red blood cell sediment at the bottom of the wells in all cases 8 to J (if the measurement is good, the negative particles are small and clear).

■ Test results For the test solution, the amount of anti-AIDS virus antibody was determined by the highest dilution ratio at which an agglutination image was observed compared to the negative control.

Experimental example: Anti-AIDS virus antibody positive results when using red blood cells sensitized with conventional AIDS virus antigens and when using red blood cells sensitized with the anti-AIDS virus monoclonal antibody of the present invention bound to AIDS virus antigens. Antibody titers in serum (5 samples) and negative serum (2 samples) were measured by hemagglutination reaction, and the differences were subtracted.

(Preparation of sensitized blood cells by conventional method) Mix the suspension of 5% nitric acid-treated blood cells of Example 1 with an AIDS virus antigen solution (1 to 50 ng/ml), pH 7.2. The cells were left at 37° C. for 1 hour to obtain AIDS virus antigen-sensitized blood cells.The cells were thoroughly washed with phosphate buffer.The sensitized blood cells were dispensed and freeze-dried to obtain a reagent.

The measurement results are shown in Table 1.

1. Indication of the case 1986 Patent Application No. 406 Table 2. Name of the invention Method for measuring anti-AIDS virus antibodies and its measuring reagent kit 3. Person making the amendment Relationship to the case: Name of the patent applicant 2. Stocks Company Midori Juji 4, Agent address: January 5, 3-8 Kasumigaseki, Chiyoda-ku, Tokyo 100, Japan Date of amendment order: The sensitized blood cells of the present invention have achieved high sensitivity.

Agent Patent attorney (8107> Kiyotaka Sasaki (and 3 others) 6. Number of claims increased by amendment, ? Ill correct target: Column 8 of "Detailed description of the invention" in the memorandum, Contents of amendment : The "Detailed Description of the Invention" column of the specification is amended as follows.

(1) Statement box page 2, line 11, “Immunode F
iciency J r Immuno Dclcia
ncy J and correction.

(2) On page 6, lines 15-16 of the same book, "cancer cells (i.e., specific antigens with the above characteristics)" are referred to as "AIDS virus antigens"
and correct it.

(3) Line 1 from the bottom of page 9 of the same book and line 8 of page 14, r
Correct 50nl to "50μq".

(4) Delete "(boy)" from page 15, line 17 of the same book.

(5) Isomorphism, page 16, line 6, r 50119J to r50
Correct as rIjIJ.

Claims (2)

[Claims] (1) In the method of measuring anti-AIDS virus antibodies using an agglutination reaction, a sample is added after reacting an agglutination carrier sensitized with an anti-AIDS virus monoclonal antibody with an AIDS virus antigen, and the measurement amount in the sample is A method for measuring an anti-AIDS virus antibody, which comprises further reacting the anti-AIDS virus antibody. (2) (i) A sensitized carrier in which an AIDS virus antigen is bound to an agglutination carrier sensitized with an anti-AIDS virus monoclonal antibody, (ii) a positive control, (iii) a negative control, and (iv) a measurement buffer. Anti-AIDS virus antibody measurement reagent kit for agglutination reaction.