JP2895584B2 - Latex agglutination test method and reagents used therefor - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to a latex agglutination test method, and more particularly, to a method for measuring hormones, enzymes, proteins, antibodies, etc. present in blood, The present invention relates to a latex agglutination test method that does not require separation.

[Prior art and its problems] In a latex agglutination test, latex particles are coated with an antibody or an antigen against a test substance, and a test sample, for example,
This method measures the amount of antibody or antigen (test substance) in a test sample by mixing and contacting it with serum, urine, culture solution, extract, etc., and judging the resulting aggregation of latex particles with the naked eye. .

However, it has been conventionally reported that whole blood cannot be used as a test sample in the latex agglutination test method, and there has been no report of test results using whole blood as a sample.

The reason is that when whole blood is used as a sample, the aggregate of latex sinks beneath the red blood cells and its formation cannot be confirmed.
Trying to see the clumps submerged beneath the red blood cells,
For example, even when the test plate was tilted, it was not possible to discriminate between those due to natural precipitation and those due to aggregation.

For this reason, whole blood is not used as a test sample, but serum is used.To obtain serum, blood must be centrifuged or the like. The latex agglomeration method could not be used if urgent testing was needed outdoors without equipment.

[Means for Solving the Problems] The present inventors have conducted intensive studies to extend the application of the latex agglutination method to whole blood samples, and as a result, appropriately selected the specific gravity of latex particles and generated the reaction. If latex agglomerates are allowed to float on the surface of the reaction system,
It has been found that the latex agglutination method can also be applied to whole blood samples.

That is, the present invention relates to a latex agglutination test method in which an antigen or antibody in a sample is reacted with an antibody or an antigen bound thereto and a latex to which the antibody or antigen is bound, and the resulting latex agglutination measures the presence of the antigen or antibody in the sample.
An object of the present invention is to provide a latex agglutination test method using a whole blood sample containing red blood cells as a test sample and causing latex agglutination generated by the reaction to float on the surface of the reaction solution.

In the method of the present invention, in order to cause latex aggregation to float on the surface of the reaction solution, the specific gravity of the latex bound to the antibody or antigen used for the test (hereinafter referred to as “test latex”) and the liquidity of the reaction system, such as salts and proteins, Type and concentration are important. Specifically, the specific gravity of the test latex must be almost the same as or slightly higher than the specific gravity of the non-hemocyte fraction of the reaction system consisting of the whole blood sample and the latex suspension, and lower than the specific gravity of the blood cell fraction. It is.

In order to prepare such a test latex, a low specific gravity latex having a specific gravity of about 0.990 to 1.080 is used as a raw material latex, and the coating with an antibody or an antigen may be made to fall within the specific gravity range of the above conditions. The preferred specific gravity range of the test latex varies depending on the specific gravity of the measurement system and the like, and cannot be limited by a numeral, but is generally about 1.015 to 1.080.

The coating of the raw material latex with the antibody or antigen may be performed according to a conventional method, and is not particularly limited.

In the present invention, as will be described later, the specific gravity of the test latex and the latex in which the test latex is aggregated, the liquidity of the reaction system, and the specific gravity of the blood cell fraction are important.

In the latex agglutination test of the present invention, since the added reagent, salt and protein concentration differ depending on the substance to be measured, the specific gravity of the test latex and the agglomerated latex to be used and the liquid property of the reaction system should be determined experimentally. is necessary.

The specific gravity of the test latex can be adjusted by adjusting the specific gravity of the raw latex and the coating amount of the antigen or antibody on the raw latex.

The present invention can be carried out in the same manner as the known latex agglutination method, except that the test latex described above is used as the latex used in the test and a whole blood sample is used as the test sample.

That is, after the test latex is added to the whole blood sample and allowed to react, the presence of the substance to be measured may be determined from the aggregated latex floating on the surface of the reaction system.

[Action] In the latex agglutination reaction, an antigen or an antibody is coated on the surface of latex particles, and an antibody or an antigen reactive with the antigen or the antibody is measured by agglutination of the latex. The present invention is a reaction system focusing on the fact that the specific gravity of latex particles for aggregation in various reaction liquid properties is lower than the specific gravity of blood cell components and higher or equal to that of serum components.

That is, when the agglutinating reagent of the present invention is added to a whole blood sample, it forms a uniform reaction system, and latex agglomerates generated by the reaction float on the surface of the reaction solution due to buoyancy. It is based on a new technical idea of making it possible.

[Effects of the Invention] Conventionally, the latex agglutination method cannot be applied to a whole blood sample, and can be implemented only after performing serum separation. However, according to the method of the present invention, it has become possible to detect hormones, enzymes, antibodies and the like present in blood without separating serum.

Therefore, according to the present invention, not only the detection of the above components in the blood is facilitated, but also, for example, even in a situation where serum separation is difficult in the field of livestock, etc. In addition, the amount of progesterone in blood can be measured quickly, and it can be used very advantageously.

[Examples] Hereinafter, the present invention will be described in more detail with reference to Examples.
In the following examples, results of tests performed on an antibody against a mouse monoclonal antibody (hereinafter, referred to as “HAMA”) by the method of the present invention are shown. However, the method of the present invention is, of course, not limited to these substances. Absent.

The measurement of HAMA and HAMA-like substances (substances that show cross-reactivity with mouse monoclonal antibodies) in the examples is of significant significance in immunoscintigraphy and immunotherapy using mouse monoclonal antibodies.

In other words, immunoscintigraphy and immunotherapy are medical techniques that are applied multiple times to the same person. When a mouse monoclonal antibody is applied to the human body, an antibody (HAMA) against it is produced, and as a result, an immunocomplex is formed. As a result, the original effect of drug administration cannot be expected.

In addition, some rheumatoid factors and antinuclear (ssDN)
A) When an antibody is present, it shows cross-reactivity with a mouse monoclonal antibody, and the original medical effect cannot be expected like a HAMA-like substance.

Therefore, it is important in medical technology to measure the amounts of HAMA and HAMA-like substances present in the body before administering an immunoscinti agent or an immunotherapy agent.

Example 1 Preparation of low-density latex agglutinating reagent (product of the present invention): 1 ml of latex suspension (N-1000 manufactured by Sekisui Chemical Co., Ltd. (particle size: 1.00 micron, solid content: 10%, specific gravity: 1.003)) in a 50 ml centrifuge tube and 0.05 A mouse monoclonal antibody was added to 9 ml of M glycine buffer (pH 8.5, containing 0.15 M sodium chloride) at a concentration of 1 mg / ml, and the mixture was vortexed, and allowed to stand at 50 ° C. for 30 minutes. After centrifugation at 4000 rpm for 15 minutes in a high-speed centrifuge (KUBOTA RR / 20000), the supernatant was removed, and then a 0.01 M glycine buffer solution (pH 8.6, 1
The latex was resuspended in 10 ml of the mixture containing 10% BSA and 0.15 M sodium chloride. The mouth of the centrifuge tube was closed with parafilm, and the tube was allowed to stand at 4 ° C. overnight. The mixture was centrifuged at 4000 rpm for 15 minutes, and the supernatant was removed. 0.05M glycine buffer (pH 8.6,
The latex was resuspended in 10 ml of 0.05% BSA, 0.01% sodium azide, and 0.15M sodium chloride. 40
The mixture was centrifuged at 00 rpm for 15 minutes, and the supernatant was removed. This operation was repeated three times. 0.05M glycine buffer (pH 8.6, 0.05
% BSA, 0.01% sodium azide, and 0.15 M sodium chloride) (5 ml) to obtain a low-density latex agglutination reagent (latex content: 2%) solution.

Comparative Example As a latex suspension, a high specific gravity latex (SDL-59 manufactured by Takeda Pharmaceutical Co., Ltd., particle size 0.90 micron, solid content 5%)
A specific gravity latex agglutination reagent (latex content: 2%) solution (comparative product) was obtained in the same manner as in Example 1 except that 2 ml of specific gravity was used.

Example 2 Comparison of low specific gravity latex agglutinating reagent and high specific gravity latex agglutinating reagent: Using the latex agglutinating reagent prepared in Example 1 and Comparative Example, detection of HAMA was tested by the following method. Table 1 shows the results.

(1) Measurement method latex agglutination determination plate (Sekisui Chemical Co., Ltd. C-HP10) of N _o 1, 2 and low density latex agglutination reagent in the reaction zone within the frame 3 (the present invention) or high density latex agglutination 50 μl of each reagent (comparative product) is added dropwise.
In the NO.1 reaction zone frame, anti-mouse IgG affinity-purified using fresh blood collected from heparin from normal rabbits
(H + L) rabbit antibody (Kappel # 0611-0082)
50 μl of a 1000-fold dilution was added and stirred. The reaction zone in the frame N _o .2, fresh blood was heparinized blood from normal rabbit 50
μl was added dropwise, mixed with a stirring rod, and spread in the reaction zone frame. The reaction zone in the frame N _o .3, sample and 4.3m plus the N _o .1
25 μl of g / ml mouse monoclonal antibody solution (0.01 M phosphate buffer) was added dropwise, mixed with a stirrer, and spread in the reaction zone frame. The judgment plate was gently moved for 1 minute and allowed to stand still for 10 minutes, and the aggregation image was observed and judged.

(2) Measurement results As is apparent from this result, when using a low density latex agglutination reagent, latex aggregates of N _o .1 is emerge on the reaction solution surface, aggregation could be clearly determined. In contrast, clumps of N _o .1 high density latex reagent sinks to the bottom of red blood cells, was not confirmed its clumps. In addition, standing for 10 minutes caused the latex particles to sink below the red blood cells, making it impossible to determine _No. 2 or 3.

In contrast, the N _o .2 low density latex agglutination reagent 3 can check the state of unaggregated Ratekkkusu particles unaggregated is on the red blood cells, was negative determination it is also easily possible.

The photographs showing the above-mentioned states are shown as reference photographs.

Claims (3)

(57) [Claims] 1. A latex agglutination test method comprising reacting an antigen or antibody in a sample with a latex to which the antibody or antigen is bound, and measuring the presence of the antigen or antibody in the sample by the resulting latex agglutination. A latex agglutination test method, wherein a latex agglutination generated by a reaction is caused to float on the surface of a reaction solution, using a whole blood sample containing red blood cells as a sample. 2. The specific gravity of a latex to which an antibody or an antigen is bound is substantially the same as or slightly higher than the specific gravity of a non-hemocyte fraction of a reaction system comprising a whole blood sample and a latex suspension, and
The latex agglutination test method according to claim 1, wherein the specific gravity is lower than the specific gravity of the blood cell fraction. 3. An antibody or an antigen against the test sample is determined so that the specific gravity is substantially the same as or slightly higher than that of the non-blood cell fraction of the reaction system containing the whole blood sample and lower than the specific gravity of the blood cell fraction. Latex agglutination test reagent in whole blood sample containing bound latex.