JPS6022295B2 - Aqueous solvent for red blood cell agglutination test - Google Patents

Description

Detailed Description of the Invention The present invention is based on an immune reaction, in which an antigen or antibody is mixed with a specimen in an aqueous solvent, and the agglutination reaction (strength of agglutination) is followed to quantitatively measure the antigen or antibody. This relates to the aqueous solvent used when mixing an antigen or antibody with a specimen in a red blood cell agglutination test.

There is a method to quantitatively measure one or both of these by tracking the antigen-antibody reaction or the agglutination reaction based on the immune reaction between a certain type of animal, plant, bacterial, or virus-derived agglutinating substance and biological protein or red blood cells. , hemagglutination reaction (PHA)
RPHA method), reverse passive hemagglutination (RPHA method), bacterial agglutination, latex agglutination, etc., are widely used in the field of clinical testing.

For example, to explain the PHA method, sheep red blood cells are sensitized using purified HBs antigen as a carrier for an agglutination test.
When Bs anti-cool sensitized sheep red blood cells are obtained and mixed with a blood sample in an aqueous solvent, whether or not an agglutination reaction is observed can be used to determine whether HBs antibodies are present in the sample.

In addition, if agglutination is observed, quantitatively measure the Japanese and European antibodies contained in the sample by diluting the sample in stages and determining the dilution ratio until no agglutination reaction can be observed. I can do it. Next, in the RPHA method, purified H
HBs antibody-sensitized red blood cells are prepared by sensitizing the red blood cells of mammalian animals using Bs antibody as a carrier for agglutination tests, and when this and the specimen are mixed in an aqueous solvent, whether or not an agglutination reaction is observed is determined. It is possible to detect whether a sample contains HBs antigen. Furthermore, certain viruses and avian red blood cells cause the red blood cells to agglutinate in an aqueous medium due to the action of the agglutinin of the virus. The presence or absence of agglutination reaction in this aqueous solvent is examined, and the concentration of the virus can be quantitatively measured based on the strength of agglutination. Many of these red blood cell agglutination tests that utilize agglutination reactions in aqueous solvents are simple and have high measurement sensitivity, so they are widely used for testing various antigens and antibodies.

However, a common drawback of these hemagglutination tests in aqueous media is that nonspecific agglutination reactions occur in samples containing pseudo-reactive substances such as lipoproteins, fibrinogen, or partially denatured proteins. It is easy to make mistakes. When measuring such samples, either dilute them in advance to the extent that they are not affected by these pseudo-reactants, or perform the measurement using an appropriate confirmation test method to prevent the agglutination reaction in an aqueous solvent. It is necessary to check the authenticity of Therefore, it is already known that as a way to reduce nonspecific agglutination reactions in these aqueous solvents, it can be improved to some extent by adding a small amount of animal protein or soluble red blood cell membrane components to the aqueous solvent used for measurement. There is. However, since many non-specific reactions still remain, the emergence of a better improvement method has been awaited. In addition, as another method to eliminate non-specific reactions, it is unavoidable to remove these pseudo-reactive substances in the specimen by adsorbing them to kaolin, blood cells, etc. It was too complicated to handle. An object of the present invention is to provide an aqueous solvent for hemagglutination tests that eliminates nonspecific reactions in hemagglutination tests and has extremely excellent detection specificity. The inventors have continued research to improve the test method in order to eliminate non-specific reactions in hemagglutination tests based on long-term immune reactions. It was discovered that the measurement specificity was significantly improved by using a chelating agent such as the following, and the present invention was completed based on this new finding. The present invention uses BeH, MgM, sr., Ba., Fe juice,
zn days, at least one divalent gold ion selected from CdH was added at a concentration of 0.001 to 1. It is added at a concentration of M.
In addition, the present invention provides divalent metal ions in an aqueous medium for red blood cell agglutination test in an amount of 0.001 to 1. a chelating agent such as ethylenediaminetetraacetate (EDTA) at a concentration of 0.005-1. In addition to the concentration of the marking, divalent metal ions and chelating agents are used in small amounts. 0.001M or less of divalent metal ions or 0.005M of EDTA
When added at concentrations below these additives are unable to suppress non-specific aggregation and reduce divalent metal ions to 1. ■
M or more or EDTA 1. If it is added at a concentration higher than M, the sensitivity of the hemagglutination reaction will decrease. As the aqueous solvent, any known aqueous solvent for hemagglutination tests can be used, such as water, physiological saline, various buffers (phosphate buffer, Tris-HCl buffer, borate buffer, glycine buffer), albumin solution. , dextran solutions, normal human and animal serum solutions, synthetic polymer substance solutions, surfactant-containing aqueous solutions, and solutions consisting of combinations thereof.

A suitable solution for an aqueous solvent is about 4.0 to 9.0, preferably adjusting the pH with a buffer. The concentration of the salt contained in the aqueous solvent is preferably relatively low, and more preferably a physiologically isotonic solution. Note that the above-mentioned known non-Samurai agglutination inhibiting substance (animal protein or soluble red blood cell membrane component) may be mixed with this aqueous solvent. The carrier for the red blood cell agglutination test using an aqueous solvent according to the present invention is O type human or sheep;
Preferably, red blood cells from animals such as guinea pigs and chickens are fixed with formalin, glutaraldehyde, etc.

The size of the carrier is preferably about 20 r or less, and granular carriers having about 5 to 15 ridges are particularly preferred. Treatment for sensitizing such particulate carriers with antigens or antibodies is carried out according to known methods. Experiments were conducted to prove the effectiveness of the red blood cell agglutination test using an aqueous solvent according to the present invention.

First, guinea pigs were immunized with purified HBs antigen to obtain Japanese maternal antiserum, and this antiserum was treated with ammonium sulfate partitioning method and ion exchange chromatography method to obtain purified HBs antibody. Fixed sheep red blood cells are sensitized to obtain HBs antibody-sensitized sheep red blood cells. On the other hand, normal animal serum and sheep red blood cell membrane components were added to 0.1M Tris-HCl buffer (pH 7.5) to prepare a known aqueous solvent for the RPHA method, and divalent metals were added to this aqueous solvent as shown in Table 1. Ions (BeH, MgH, Sr, Ba
An aqueous solvent according to the present invention was prepared by adding 1, Fe, 2, 3, Fe, 2, 3, Cd juice) alone, and as a control, the one without additives was shown in the column of MgC12 in Table 1 as the amount added for each sample as 0. , and the addition amounts of 0.0001M and 1.9M are shown in the same column.

According to the RPHA method, three samples (No. 1, No. 2, and No. 3 in the table) of citrate-induced blood elutriation, which are known to cause non-specific agglutination as a result of confirmation tests using conventional methods, were Two-fold dilutions were performed using these three types of solvents on a microplate, and the above-mentioned HBs antibody-sensitized sheep red blood cells were mixed in the dilution series. Separately, a specimen (No. 4) positive for HBs antigen was similarly treated and tested. The amount of mixed liquid is 25 μg of sample diluent, 25 μg of HBs antibody-sensitized sheep red blood cells.
The strength of aggregation (degree of aggregation) after 2 hours at room temperature was evaluated as 4, 3, 2, or 1, with non-aggregation being evaluated as 0, and the results shown in Table 1 were obtained. Table 1 In the case of divalent metal ions alone, as can be seen from Table 1, non-specific aggregation of 1:16 to 1:32 occurs in an aqueous solvent without divalent metal ions, whereas in a solvent with an appropriate amount of divalent metal ions added. The degree of aggregation was suppressed to 1:1 to 1.8, and even when 0.0001 M of MgC12 was added, the degree of aggregation was the same as that without addition, and nonspecific aggregation was not suppressed.

In addition, specimen No. 1 positive for HBs antigen. 4 shows that even when an appropriate amount of divalent metal ions is added, it is enteric, indicating that this addition does not affect positive samples, and MgC12 was added to 1.0M.
No decrease in sensitivity was observed even if the addition amount was 1. When it comes to shoes, the sensitivity decreases, indicating that the amount added is too high. Next, when two types of divalent metal ions were added to a known aqueous solvent in the RPHA method, and one type of divalent metal ion and ethylenediaminetetraacetic acid trisodium salt (EDTA)
The same experiment as in the case of divalent metal ion alone was conducted for the case where both - and a) were added, and the results shown in Tables 2 and 3 were obtained, respectively.

Table 2 Table 3 when two types of divalent metal ions are used together
When divalent metal ions and EDTA are used in combination, as can be seen from Table 2, non-specific aggregation is suppressed to a ratio of 1:1 by using two types of divalent metal ions in combination. No decrease in sensitivity was observed in Sample No. 4. Also, as can be seen from Table 3, by using 0.008M and 0.08M EOTA-Funa a together, non-specific aggregation was suppressed to 1:1 to 1:2, but the amount added was 0.001M. In the case of Mg
It is no different from the case of C12 alone. Also, sample no. In 4, EDTA- a is 1. (2) Even when M is added, no bottom drop in sensitivity is observed, but when the amount added exceeds 1.9 M', the sensitivity decreases, indicating that the amount added is too large. According to the present invention, it is possible to obtain an aqueous solvent that can significantly suppress non-specific agglutination in a red blood cell agglutination test between an antigen or antibody and a specimen based on an immune reaction, and it is used in the measurement of various antigens and antibodies. This has the effect of simplifying the hemagglutination test and significantly increasing detection non-specificity.

Examples of the present invention will be described below.

Example 1 Magnesium chloride (MgCl21 fine 20) 40 saw PH
7.6 0.1M Tris-HCl buffer is added to prepare 2000 volumes of aqueous solvent for red blood cell agglutination test.

Example 2 Physiological saline was added to 1.22 hours of barium chloride (BaC12, 2 days and 2 hours) and 2 hours of healthy rabbit serum to prepare 200 hours of an aqueous solvent for red blood cell agglutination test.

Example 3 A 200% aqueous solvent for red blood cell agglutination test was prepared by adding 1.0 g of zinc acetate {Zn(CH3COO)2.20} and 2 portions of healthy rabbit serum to physiological saline.

Example 4 Magnesium chloride 4.0 and ethylenediamine 4 acetic acid 3
An aqueous solvent for hemagglutination test is prepared by adding 0.1M phosphate buffer, pH 7.6, to 5.09% sodium salt and 20% bovine serum albumin.

Example 5 Barium chloride (BaC12.2LO) 5.0% and ethylenediaminetetraacetic acid trisodium salt 5.0% and 20%
A 0.1M phosphate buffer solution with a pH of 7.6 is added to 10% of bovine serum albumin to prepare 200% of an aqueous solvent for a hemagglutination test.

Claims (1)

[Claims] 1. In a hemagglutination test in which an antigen or antibody and a specimen are mixed in an aqueous solvent based on an immune reaction, and the antigen or antibody is measured by tracking the agglutination reaction, Be^+^+, Mg^
+^+, Sr^+^+, Ba^+^+, Fe^+^+,
At least one selected from Zn^+^+, Cd^+^+
An aqueous solvent for a red blood cell agglutination test, characterized in that it contains two divalent metal ions at a concentration of 0.001 to 1.0M. 2 In the red blood cell agglutination test, in which antigens or antibodies and specimens are mixed in an aqueous solvent based on immune reactions, and the antigens and antibodies are measured by tracking the agglutination reaction, Be^+^+, Mg^
+^+, Sr^+^+, Ba^+^+, Fe^+^+,
At least one selected from Zn^+^+, Cd^+^+
divalent metal ions at a concentration of 0.001 to 1.0M, and ethylenediaminetetraacetate at a concentration of 0.005 to 1.0M.
．． An aqueous solvent for a red blood cell agglutination test, characterized in that it is contained at a concentration of 0M.