JPS6147566A - Antirubella virus antibody sensitized corpuscle and measurement of antirubella virus antibody - Google Patents

Abstract

PURPOSE:To measure antirubella virus antibody free from action of an inhibiter depending on an reversibly passive corpuscle agglutination blocking reaction (RPHA-HI), by employing an antirubella virus antibody sensitized corpuscle obtained by bonding an antirubella virus antibody to red corpuscle through a coupling agent. CONSTITUTION:The red corpuscle used for a sensitized corpuscle is preferably an immobilized red corpuscle viewed from preservability. The immobilized red corpuscle is produced by treating raw red corpuscle suspended in an alsever solution by carbon monoxide gas to immobilize it with addition of formalin thereto. The red corpuscle is treated using a coupling agent to obtain a corpuscle having the coupling agent bonded to the surface thereof and a liquid containing an antirubella virus antibody is brought into contact with the corpuscle thus obtained to create an antirubella virus antibody sensitized corpuscle. The coupling agent employs tannic acid. A rubella virus corpuscle agglutinin (HA) is added to a sample such as human serum or the diluted liquid thereof and then, the antibody sensitized corpuscle is brought into contact with the mixture. If any antibody exists in the sample, agglutination will not occur, where the blocking of corpuscle agglutination is observed.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to anti-rubella virus antibody-sensitive blood cells and a method for measuring anti-rubella virus antibodies using the same.

[Prior art and highlights]

Rubella virus is an RNA virus similar to Togavirus that is transmitted from person to person and causes rubella.
As a result, the rubella that develops is relatively mild. On the other hand, it is well known that if a pregnant woman becomes infected during the early stages of pregnancy, it will cause serious damage to the fetus, resulting in a high rate of congenital abnormalities.

However, once infected with this virus, a person acquires lifelong immunity and is protected from reinfection. Therefore, determining the history of infection,
Measurement of immune antibodies is essential for determining whether or not vaccination is appropriate.

Currently, the measurement of this antibody requires rubella virus hemagglutinin (
Hemagglutination inhibition reaction (hereinafter referred to as rHI reaction), which measures antibodies that inhibit hemagglutination caused by rHAj)
) is widely applied. However, since inhibition of hemagglutination in this reaction occurs not only by antibodies but also by insulators and bitters in the blood, the inhibitors must be removed in advance. However, removal of this inhibitor not only requires complicated operations, but also makes it impossible to judge whether it has been removed or not. Therefore, when a low agglutination inhibition value is shown, it is difficult to determine whether the inhibitor is the antibody or whether there is residual inhibitor. It has the disadvantage that it is not possible to reliably determine whether

[Failure to resolve the gap]

The present inventor first purified HA, immunized a rabbit with it to produce an antibody against HA, sensitized this antibody to blood cells using a coupling agent, and performed a reverse passive hemagglutination reaction (hereinafter referred to as r).
We have developed a new method to detect IHA by RPHA J). Next, 'This method is used to determine whether HA added to a sample such as patient serum has been neutralized by antibodies in the sample, that is, reverse passive hemagglutination inhibition reaction (hereinafter referred to as RPHA-HI j).
) established a method for measuring antibodies against HA in a sample, and completed the present invention. This method does not involve any inhibitor at all, so there is no need to remove the inhibitor at all, and it has the great advantage of being able to specifically measure the anti-hemagglutinin antibody titer (hereinafter abbreviated as rAHA titer).

Conventionally, neither RPHA for HA nor RPHA-HI for measuring the AHA titer was known, and it was impossible to determine the HI titer without an inhibitor removal operation, but with the present invention, there is no inhibitor removal operation. It became possible to specifically measure the AHA titer, which is equivalent to the HI titer.

The object of the present invention is to use anti-rubella virus antibody-sensitive blood cells, which are made by binding anti-rubella virus antibodies to red blood cells via a coupling agent, as well as anti-rubella virus antibodies by RPHA-HI without the involvement of an inhibitor. An object of the present invention is to provide a method for measuring rubella virus antibodies.

The red blood cells used for the sensitive blood cells of the present invention are live red blood cells or fixed red blood cells, but it is preferable to use fixed red blood cells from the viewpoint of preserving the sensitive blood cells. Live red blood cells are obtained from sheep, goats, cows, horses, guinea pigs, chickens, turkeys, humans, etc. according to conventional methods. Fixed red blood cells are prepared as follows. That is, Alseper (, Al
Live red blood cells suspended in 5ever) solution are treated with carbon monoxide gas, and formalin is added to the red blood cells to perform fixation.

As the coupling agent, tannic acid, glutaraldehyde, chromium chloride, etc. can be used, and among them, tannic acid is preferred.

Rubella virus HA can be easily obtained by culturing tissues such as BHK cells. That is, BHK cells are cultured in a monolayer, infected by inoculation with rubella virus, and the culture supernatant is collected when the virus has sufficiently proliferated.

This supernatant was cooled to 4°C, and goose blood cells cooled to 4°C were added to it to sufficiently adsorb HA. After centrifugation at 4°C to separate the blood cells, this was added to physiological saline. When suspended and humidified at 37°C, HA is released from blood cells, so
Centrifuge and collect the supernatant fraction. Collect the supernatant and centrifuge 4
Anti-HA antibodies can be easily obtained by centrifuging at 0.000 rpm for 60 minutes, collecting the precipitate, and immunizing rabbits, goats, etc. according to conventional methods.

The sensitive blood cells of the present invention are produced as follows. That is, red blood cells are treated with a coupling agent to obtain coupling agent-treated blood cells (red blood cells with a coupling agent bound to the surface of the red blood cells), which are then brought into contact with a solution containing anti-rubella virus antibodies to produce anti-rubella virus antibodies. Obtain antibody-sensitive blood cells.

As a diluent, use a warm canned glycine saline solution, phosphate buffered saline solution, etc. to which approximately 0.1% bovine serum albumin (hereinafter abbreviated as "BsA") is added, and 0.05 to 0.5%. Add sodium azide (NaN, ).

The sensitive blood cells thus obtained may be suspended in a diluent to a concentration of about 2.5% by weight and stored in an ice chamber, or may be freeze-dried. For freeze-drying, add 0.2 each of various amino acids, especially glycine or monosodium glutamate, and dextran as stabilizers to the diluent.
~2% by weight and 0.3~3% by weight are added, rapidly frozen in liquid nitrogen or liquid air, and then freeze-dried.

Freeze-drying further extends the shelf life and is usually stable for more than two years.

Since the anti-rubella virus antibody-sensitive blood cells of the present invention are agglutinated by HA, first, a certain amount of HA is added to a sample such as human serum or a diluted solution thereof, and the HA is neutralized by leaving it at room temperature for about 30 minutes. When antibody-sensitive blood cells are brought into contact, the added HA will cause an agglutination reaction if the antibody is not present in the sample or its diluted solution, but on the contrary, if the antibody is present in the sample or its diluted solution, the added HA will cause an agglutination reaction. Since HA is neutralized, agglutination does not occur even if the antibody-sensitive blood cells are added thereto. In other words, inhibition of hemagglutination is observed. When this reaction is carried out by the microtiter method, it can be observed on the microplate as an aggregated image or a non-aggregated image at the bottom of the tube. That is,
A predetermined amount of the diluent is dispensed dropwise onto a plate, and then a predetermined amount of a sample such as serum that has been absorbed with blood cells to a large extent is added and sequentially diluted with a diluter. After adding a certain amount of HA and mixing thoroughly, the antibody-sensitive blood cells are dispensed dropwise after being left at room temperature for about 20 to 60 minutes, and the presence or absence of an agglutinated image at the bottom of the tube is determined after a certain period of time. The antibody titer is the reciprocal of the maximum dilution factor of the sample that inhibits agglutination. That is, the pretreatment of the sample consists only of blood cell absorption, and the operation is extremely easy and simple, and no special techniques are required. Moreover, since no inhibitors are involved, this measurement method is specific and
Sensitivity can be controlled by adding HA,
It is also easy to make the sensitivity higher than the HI reaction of the sermon.

Furthermore, it is possible to perform qualitative or quantitative analysis of a large number of analytes at the same time. Currently, RPHA fl against rubella virus is completely unknown, and no method for measuring anti-rubella virus antibodies using antibody-sensitive blood cells has been found in the literature, and the RPHA-HI reaction of rubella virus is completely new.

[Embodiments of the invention]

Next, the present invention will be explained in more detail with reference to Preparation Examples and Examples, but the present invention is not limited by these unless the gist of the invention is exceeded.

Preparation Example 1 Preparation of Guirizin Virus HA BHK21 cells were cultured in Eagle's MEM medium, and when they formed a sufficient monolayer, they were inoculated with rubella virus M33 strain.

The medium was exchanged every day for 14 days starting 3 days after inoculation, and the medium was concentrated and dialyzed against polyethylene glycol 6000 to prepare an approximately 50-fold concentrated solution. 5,000 rp
Contaminants were removed by centrifugation at m for 30 minutes to obtain a crude virus sample. This sample was dialyzed against MI and Ca-added physiological saline, cooled to 4°C, goose blood cells cooled to 4°C were added thereto, and HA was adsorbed at 4°C for 60 minutes. The blood cells were collected by centrifugation at 3,000 rpm for 10 minutes at ℃. This was suspended in physiological saline at 37°C.
The virus was heated in a thermostatic water bath for 60 minutes to liberate HA, which was then centrifuged in an ultracentrifuge at 40,000 rpm for 60 minutes to collect rubella virus, which was suspended in physiological saline and used as an HA antigen.

Preparation Example 2 Preparation of Anti-HA Antibody The LA (from Preparation Example 1) was diluted 1:10 with physiological saline, and this HA was injected into the ear vein of about 2.5 U of rabbits for 1.0 d at a time. Immunization was carried out with 10 injections every 2 days, and one week after the last immunization, whole blood was collected from the carotid artery, serum was separated according to the standard method, and anti-HA was heated at 56°C for 30 minutes.
Obtained antibodies.

Preparation Example 3 Preparation of fixed red blood cells Fresh blood obtained from chickens was added with
ever) solution and add 3.5 liters of CO gas in a walasf.
After soaking for 0 minutes, immediately add a fixative solution [physiological saline containing 5% sodium citrate + 37% formalin (volume ratio 29:1)] and leave in an incubator at 37°C for 24 hours. However, during this time it was shaken occasionally. Thereafter, it was washed five times with pure water and five times with physiological saline. Fixed red blood cells were suspended in -7.2 phosphate buffered saline containing 0.1% NaN3 to a concentration of 10% and stored in an ice chamber. This fixed red blood cell was stable for over 1 year.

Example 1 Preparation of Sensitive Blood Cells Phosphate buffered saline containing 2.5% fixed red blood cells obtained in Preparation Example 3 (CPH7,2) (hereinafter referred to as rPBs)
J) to 1:100,000 tannic acid/P
The cells were treated with tannic acid at 37° C. for 15 minutes with an equal amount of BS, washed once with PBS, and suspended in the original amount of PBS to prepare a tannic blood cell solution.

Add the anti-HA antibody obtained in Isoelectric Preparation Example 2 to this tannin blood cell solution, treat it at 37°C for 30 minutes, and then add BSA to PBS (pH 7.2) once.
It was washed once with a diluted solution containing 1% O and 0.1% NaN, and suspended in the diluted solution.

These sensitive blood cells agglutinated with rubella virus HA on a microplate, but did not agglutinate even when normal antigen prepared from virus-uninoculated cells was added. Furthermore, tannin blood cells that are not sensitive to antibodies did not aggregate even when HA was added under the same conditions. In other words, it was confirmed that these sensitive blood cells specifically reacted to rubella virus HA and agglutinated.

Example 2 Reverse passive hemagglutination inhibition reaction was carried out by the microtiter method, and first, the diluted solution was dispensed onto a plate in 0.025 mJ portions using a dropper. 1:4 absorption in the 1st 6th eye [111'ffJ 0.025
Added m7. Diluted 2N with a diluter.

4 agglutination units of I (A) were dispensed in 0.025 ml portions, mixed thoroughly, and left to stand at room temperature for 30 minutes. To this, the antibody-sensitive blood cells of Example 1 were added dropwise in 0.025 ml portions. ,
After mixing thoroughly, the image of agglutination was determined by leaving it at room temperature overnight, and the antibody titer was calculated as the reciprocal of the maximum dilution of the serum that inhibited agglutination. The measured results were as follows.

As described above, this reaction exhibited sensitivity 2 to 4 times that of conventional HI. In addition, the cases in which the II reaction was negative also showed negative results in the main reaction. In other words, it was found that the sensitivity was higher than that of the conventional HI reaction, and that it did not show non-specific positivity.

〔Effect of the invention〕

If the sensitive blood cells of the present invention are used, anti-rubella virus antibodies can be extremely easily quantified with a small amount of sample of about 0.0251 Ll by simply absorbing the sample into blood cells, so it can be said that it is particularly suitable for clinical diagnosis.

Claims (5)

[Claims] (1) Anti-rubella virus antibody-sensitive blood cells comprising an anti-rubella virus antibody bound to red blood cells via a coupling agent. (2) The sensitive blood cell according to claim 1, wherein the red blood cell is a fixed red blood cell. (3) The sensitive blood cell according to claim 1 or 2, wherein the coupling agent is tannic acid. (4) The sensitive blood cell according to any one of claims 1 to 3, wherein the anti-rubella virus antibody is an anti-rubella virus hemagglutinin antibody. (5) After adding rubella virus antigen to the sample, anti-rubella virus antibody-sensitive blood cells, which are made by binding anti-rubella virus antibodies to red blood cells via a coupling agent, are applied to the sample to cause a reverse passive hemagglutination inhibition reaction. A method for measuring an anti-rubella virus antibody, the method comprising measuring the anti-rubella virus antibody titer of .