JPS6057255A - Aqueous solvent for testing agglutination - Google Patents

Abstract

PURPOSE:To suppress the non-specific reaction by a rheumatic factor and to determine exactly quantitatively an antigen, antibody, etc. by using an aq. solvent contg. thermally degenerated IgG for various agglutination tests. CONSTITUTION:An aq. soln. contg. about 0.015-7.5mg/l IgG, more preferably, IgG originated from man which is degenerated by heating at 40-70 deg.C for 15min -3hr is used as a solvent for immunoagglutination test for preparing physiological salt soln., various buffer solns., albumin soln., serum soln., etc. The generation of the non-specific agglutination by the rheumatic factor in the sample serum, etc. is suppressed by using such solns. Detection sensitivity is thus improved and the exact measured value is obtd. in the agglutination reaction occuring in the antigen-antibody reaction. The aq. soln. of the heated IgG has a good shelf life and no difference is given to the sensitivity, etc. even if the soln. preserved for one month at 40 deg.C is used.

Description

[Detailed description of the invention] The present invention relates to an aqueous solvent for aggregation tests.

Antigen, antibody,
Methods for quantitatively measuring viruses etc. are hemagglutination reaction (PHA method) and reverse passive hemagglutination reaction (RPHA method).
, bacterial agglutination reaction, latex agglutination reaction, etc. are widely used in the field of clinical testing. for example,
To explain the PHA method, purified HBs antigen is used as a carrier for an agglutination test to sensitize sheep red blood cells to obtain HBs antigen-sensitized red blood cells, and when this is mixed with a blood sample, an agglutination reaction is observed in an aqueous solvent. Whether or not HBS antibodies are present in the specimen can be determined based on whether or not HBS antibodies are present in the specimen.

In addition, if an agglutination reaction is observed, the HBs antibody in the sample can be quantitatively measured by further diluting the sample in stages and determining the dilution ratio until the agglutination reaction can no longer be observed. .

In addition, in the R P HA method, purified H 13 S antibody is used as a carrier for an agglutination test to sensitize mammalian red blood cells to HB.
It is possible to detect whether or not the sample contains HBs antigen by preparing s-antibody-sensitized red blood cells and whether or not agglutination is observed in an aqueous solvent when the red blood cells are mixed with the sample.

Furthermore, certain viruses and avian red blood cells agglutinate red blood cells in an aqueous medium due to the action of the agglutinin of the virus. The concentration of the virus can be quantitatively measured by the presence or absence and strength of the agglutination reaction in this aqueous solvent. Many of these analytical methods that utilize aggregation reactions in aqueous solvents are
It is well known that this method is widely used for testing various antigens, antibodies, etc. because it is simple and has high measurement sensitivity.

However, depending on the type of antigen or antibody to be sensitized, a clear agglutination reaction may not be observed or no agglutination reaction may be observed in these aqueous solvents even though the agglutination test carrier has been sufficiently sensitized. It may show specific agglutination. The factor responsible for this is rheumatoid factor.

An object of the present invention is to provide an aqueous solvent for agglutination tests that exhibits an effect of suppressing nonspecific aggregation caused by rheumatoid factors and has extremely excellent detection sensitivity.

The inventors continued their improvement research to suppress the non-specific agglutination reaction caused by rheumatoid factor in this agglutination reaction, and found that by adding a small amount of heat-denatured 1gG to the measurement system, the non-specific reaction caused by rheumatoid factor was significantly suppressed. The present invention was completed based on this discovery.

That is, the present invention provides an aqueous solvent for agglutination tests based on immune reactions, which is characterized by containing heat-denatured 1gG.

By using this aqueous solvent in the preparation of various agglutination reaction test reagents, non-specific agglutination reactions are suppressed and the above-mentioned measurement sensitivity is improved.゛Heat denaturation 1 used in the present invention
As gG, a heat-denatured human-derived immunoglobulin is preferable. There are no particular limitations on the human-derived immunoglobulin, and for example, widely available commercially available immunoglobulins may be used.

The heating temperature in thermal denaturation is usually about 40 to 70°C. The heating time varies depending on the heating temperature, etc., and in general, it is preferable to shorten the heating time when the temperature is high, and to shorten the heating time when the temperature is low. It is preferable to select

The amount of heat-denatured 1gG added is generally 0.015 to 7
．． About 5 mg/ml, preferably 0.1 to 1 mg
/ml. The lower limit is the limit value of the non-specific reaction suppressing effect, and the upper limit is the limit value of the decrease in detection sensitivity.

As the aqueous solvent, any conventionally known aqueous solvent for agglutination tests can be used, such as water, physiological saline, various buffer solutions (
Phosphate buffer, Tris salt rlI buffer, borate buffer,
Glycine buffer solution), albumin solution, normal human and animal serum solutions, solutions of synthetic polymer substances, surfactant-containing aqueous solutions, and solutions consisting of combinations thereof are exemplified. The pH of the aqueous solvent of the present invention is preferably about 6.5 to 9, and is preferably adjusted using a buffer.

The concentration of the salt contained in the aqueous solvent of the present invention is preferably a relatively low concentration, for example, about 0.01 to 1%,
More preferably, it is a physiologically isotonic solution.゛As carriers for aggregation tests in which the solvent of the present invention is used, those known per se may be used, such as latex resins, rubbers, inorganic adsorbents, and particularly preferred carriers include animals (such as sheep, guinea pigs, type 0). Examples include red blood cells from humans (humans, chickens, etc.) fixed with formalin, glutaraldehyde, etc. It is advantageous to use such a carrier in the form of particles of about 20 microns or less, particularly about 5 to 15 microns. The treatment for sensitizing such particulate carriers with antibodies or antigens can be carried out by a method known per se or a method analogous thereto.

The mechanism of inhibition of non-specific aggregation in the agglutination test using the aqueous solvent of the present invention is thought to be as follows: Rheumatoid factor binds to denatured 1gG in serum or to the large excess of normal 1gG present in serum. However, when mixed with antibody-sensitized blood cells, part of the normal 1gG bound to rheumatoid factor is partially replaced with animal-derived 1gG on the surface of the sensitized blood cells, resulting in non-specific aggregation. It is imagined.

In such cases, when heat-denatured 1gG is added to the measurement buffer, most of the normal 1gG bound to rheumatoid factor is replaced with denatured 1gG, since rheumatoid factor and heat-denatured 1gG have a high affinity. It is presumed that it does not bind to animal-derived IgG on the surface of sensitized blood cells, making it difficult for non-specific aggregation to occur.

Figures 1 to 3 are diagrams showing this mechanism. In Figure 1, rheumatoid factor in blood is bound to heat-denatured 1gG, but the excess binding group is also bound to normal 1gG. The second case is when horse IgG sensitized red blood cells are mixed with this.
FIG. 3 shows normal 18G and horse IgG partially replacing each other, resulting in some non-specific aggregation. However, some rheumatoid factors only replace horse IgG in acid buffers and do not participate in aggregation.

Figure 3 is a diagram when heat-denatured 1gG is added to what is shown in Figure 1. Even if horse IgG-sensitized red blood cells are mixed with this, the addition of heat-denatured 1gG, which has a high affinity for rheumatoid factors, causes rheumatoid arthritis. The factor is saturated by heat-denatured IgG and hardly participates in non-specific aggregation.

The aqueous solvent for agglutination tests according to the present invention thus provided suppresses non-specific agglutination reactions caused by rheumatoid factors, and therefore is effective against diseases that inhibit abnormal 18G synthesis due to disturbances in the IgG synthesis system associated with various types of liver function decline. It is useful as a reagent for the system. For example, AFP.

HBsAg, Anti-11Bs+llBeAg,
It is useful as a detection reagent for Anti-HBe and the like.

Experimental example 1 AFP value is 20 by radioimmunoassay method (RIA method)
Serum samples with a concentration of ng/ml or less (serum from patients with liver disease)
Of the 1684 samples, 543 were rheumatoid factor positive.
(32.2%). When these 543 samples were measured using a conventional measurement buffer to which heat-denatured 1gG was not added, non-specific agglutination reactions due to rheumatoid factor were observed in 52 samples (9.6%).

On the other hand, when 543 samples were measured using a measurement buffer containing heat-denatured 1 gG (provided in Example 1), 10 samples still showed a non-specific agglutination reaction. Therefore, the nonspecific reaction suppression rate by using the aqueous solvent for the agglutination test is (52-1
0152) X100%='80.8%.

Experimental Example 2 Of the 766 serum samples from healthy individuals whose AFP value was 10 ng/ml or less by RIA, 81 samples (10.6%) were rheumatoid factor positive. When these 81 samples were measured using a conventional buffer solution (without the addition of heat-denatured 1gG), non-specific agglutination reactions due to the RA factor were observed in 6 samples (7.4%). When the 81 samples were measured using a heat-denatured measurement buffer containing 1 gG (provided in Example 1), 0 samples showed non-specific agglutination. Therefore, the non-specific reaction suppression rate by using the aqueous solvent for the agglutination test is 100%.

Experimental Example 3 The storage stability test of the heat-denatured 1 gG-added measurement buffer obtained in Example 1 was performed by an accelerated stability test by storage at 40°C, and it was found to be stable for 3 months at 40°C.

As a result, compared to the storage conditions where the sensitivity of antibody-sensitized animal (sheep) blood cells, which is the main component of the agglutination test reagent kit, is slightly lowered, that is, storage at 40°C for 1 month, the present invention It was found that the aqueous solvent for inventive flocculation tests exhibits higher stability.

Example 1 Phosphates, salt, animal (sheep) stroma, animal (
Separately, add an amount equivalent to 750 cm (5; l11
) heated at 56°C for 2 hours, and add 1,
000 ml to obtain an aqueous solvent for aggregation test.

Globulin Midori for intramuscular injection, buffer for measurement, 3 each
Regarding 0ft, the influence of the loft difference amount was investigated, but no difference was observed in the non-specific aggregation suppressing effect due to the loft difference.

Example 2 An aqueous solvent for an aggregation test having the following composition was obtained in one vial, containing 50 ml of sterilized and filtered isotonic phosphate buffer solution (pH 7.2) with added sodium chloride. As modified 1gG,
Venoglobulin I (manufactured by KK Midori Juji) at 40℃
The mixture was heated for 3 hours and then added.

(Composition) Disodium phosphate (anhydrous > 395mg Potassium phosphate 155mg Sodium chloride 225■ Stroma (sheep) 3% Animal serum (sheep) 1% Sodium azide 50 Akebono denatured 1gG 250mg Example 3 Globulin for intramuscular injection as denatured IgG -Midori, 70℃
Example 2 was repeated, except for 15 minutes of treatment. Experimental Example 1 was repeated using this buffer solution to obtain an aqueous solvent for the aggregation test. As a result of conducting the same test as in Experimental Example 1 using this aqueous solvent, the nonspecific reaction suppression rate by using the aqueous solvent for the aggregation test was 100%.

[Brief explanation of the drawing]

FIGS. 1 to 3 schematically show the mechanism of suppressing the non-specific aggregation reaction of the aqueous solvent for the agglutination test of the present invention. ■... Normal human IgG 2... Rheumatoid factor 3... Sensitized sheep red blood cells 4... Horse IgG (anti-AFP antibody) 5... Denatured human IgG Procedure'? lt official document (Own ship 1, Indication of the case, Patent Application No. 166931 filed in 1982, 2, Name of the invention, Aqueous solvent for flocculation test 3, Relationship with the case by the person making the amendment. Name of patent applicant: Midorijyu Co., Ltd. Imo 4, Agent 541 Address: 406 New Life Hirano-cho, 4-53-3 Hirano-cho, Higashi-ku, Osaka Telephone: (06) 227-1156 6. Number of inventions increased by amendment 7. Subject of amendment. (1) On page 4 of the specification, line 1 O, ``Uchicho Shiiku'' was corrected to ``Koichishiki''. ``
0 (3) On page 7 of the Circular, the third line is corrected to ``acidic acid value t2 < J ``phosphate buffer.'' J-

Claims (1)

[Claims] An aqueous solvent for an agglutination test based on an immune reaction, characterized by containing heat-denatured 1gG.