JPH08160044A - Method and reagent for measuring rheumatic factor - Google Patents

Abstract

PURPOSE: To enable measurement of rheumatic factor(RF) in a measuring sample with high specificity and high sensitivity by a method wherein an antigen-antibody reaction of an antigen to RF with an antibody reagent reacting with this antigen specifically is obstructed by the RF in the measuring sample. CONSTITUTION: When RF in a serum specimen and an antigen corresponding thereto are brought into an antigen-antibody reaction, the portion in the antigen which reacts specifically with the RF is blocked in proportion to the amount of the RF in the specimen. When a carrier particle sensitized with an antibody is added to this antigen, the antibody on the surface and the portion of the specific reaction in the antigen which is not blocked by the RF react with each other and an agglutination reaction occurs. In the case where the RF is very little, the portion of reaction in the antigen reagent does not decrease nealy at all in this reaction and it is agglutinated completely in reaction with the antibody-sensitized particle. In the case where the amount of the RF is large, the portion of reaction of the antigen reagent almost ceases to exist and the reaction with the antibody-sensitized particle lessens remarkably. Since the reaction of the antigen reagent with the antibody-sensitized particle is obstructed in proportion to the amount of the RF in the specimen in this way, the RF in a very small amount in the specimen can be measured with high sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunological assay method and assay reagent.

[0002]

Rheumatoid factor (RF) is a factor mainly found in the serum of patients with rheumatoid arthritis.
Reacts with IgG in human globulin. RF is known to react more strongly with partially denatured IgG,
A method of knowing the presence of RF is generally used by immobilizing IgG (modified IgG) on blood cells or latex particles and detecting the aggregation of carrier particles resulting from the reaction with RF in a sample.

[0003]

However, in the RF measurement methods currently used, many diseases other than rheumatoid arthritis, such as systemic lupus erythematosus, collagen diseases such as systemic sclerosis, chronic active hepatitis, It has a high positive rate in liver diseases such as cirrhosis and various infectious diseases, and has a problem of low disease specificity for rheumatoid arthritis.

Other than RF, as substances that react with human IgG, anti-Gm factors that react with Gm factor, which is a cognate antigen on IgG molecules, Milgrom anti-antibodies that are said to react with the antigen binding site of IgG, and IgG It is known that there is a pepsin agglutinator that reacts with the binding site generated by pepsin degradation.

Further, as mentioned above, anti-IgG antibodies found in autoimmune diseases and anti-Ig antibodies which are considered to be caused by immune complexes between bacteria and antibodies against them in various infectious diseases.
It is considered that the presence of G antibody and the like is one of the factors that reduce the disease specificity of RF.

[0006]

The present invention has been made to solve the above-mentioned conventional problems and to measure an anti-IgG antibody having high RF specificity as true RF. In the method for measuring RF and the measuring reagent, the antigen-antibody reaction between the antigen corresponding to RF and the antibody reagent that specifically reacts with the antigen is blocked by RF in the measurement sample. is there.

As the antigen corresponding to RF, IgG purified from serum of patients with rheumatoid arthritis, denatured human or rabbit IgG, or a degradation fragment thereof, and synthetic antigen corresponding to the epitope recognized by RF can be used as they are. Although it is also possible, carrier antigens may be sensitized with these antigens for higher detection sensitivity.

As the antibody reagent which specifically reacts with the above-mentioned antigen, an antibody isolated and purified from the serum of a patient with rheumatoid arthritis and an Ig purified from the serum of a patient with rheumatoid arthritis are used.
A monoclonal antibody prepared using G as an antigen may be used,
Further, polyclonal antibodies obtained by fractionating antisera obtained by immunizing animals such as rabbits and goats with these antigens may be used.

As the carrier particles, synthetic resin particles such as polystyrene latex particles and high specific gravity latex particles, synthetic particles such as gelatin, red blood cells, liposomes, metal colloid particles and the like can be used. These carrier particles can also be used as carrier particles for an antigen corresponding to the antibody to be measured.

As a method for sensitizing carrier particles with a monoclonal antibody or other polyclonal antibody, or an antigen corresponding to an antibody to be measured, general physical adsorption, or chemistry with a water-soluble carbodiimide or a bifunctional reagent is used. The dynamic binding method is used.

In a reaction system utilizing red blood cells, high specific gravity latex particles, gelatin particles, etc., the reaction result can be visually determined from the sedimentation image of the carrier particles. Further, in a reaction system using metal colloid particles as a carrier, it is visually judged from the color tone change of the colloid solution. Furthermore, it is also possible to measure by a technique of immunochromatography.

On the other hand, in a reaction system using latex particles or liposome particles of 0.05 to 1.6 μm as a carrier, the presence or absence of particle aggregation can be visually determined on the reaction plate. It is also possible to make an optical quantitative measurement by utilizing.

As the measuring method, in addition to the above-mentioned immunoturbidimetric method or carrier agglutination reaction method, existing measuring methods such as radioimmunoassay and enzyme immunoassay can be widely applied. Radioisotopes, enzymes, chemiluminescent compounds and the like can be used.

As the sample containing RF, body fluids such as serum, plasma, blood, ascites, ascites, synovial fluid and the like are used.

[0015]

[Function] When an RF-positive serum sample is reacted with an antigen composed of heat-denatured human IgG, the RF in the sample and the heat-denatured human IgG cause an antigen-antibody reaction, which is proportional to the amount of RF in the sample. The RF-specific reaction site in heat-denatured human IgG is blocked.

Next, when carrier particles sensitized with a monoclonal antibody or a polyclonal antibody for determination of an RF-corresponding antigen are added, the antibodies on the surface of the carrier particles and the specific reaction portion of the heat-denatured human IgG which is not blocked by RF. React with each other to cause an agglutination reaction.

This agglutination reaction occurs in inverse proportion to RF in the sample. That is, when RF is very small or absent, the reaction sites of the antigen reagent hardly decrease, and thus the reaction with the antibody-sensitized particles causes complete aggregation.

When the carrier particles are erythrocytes, gelatin particles, etc., they are visually judged as an agglutination image with no settling ring. When the carrier particles are latex particles, the turbidity becomes maximum and the difference in absorbance becomes maximum.

When the amount of RF is large, almost no reaction site of the antigen reagent remains, and the reaction with the antibody-sensitized particles becomes extremely low. Therefore, when the carrier particles are red blood cells, gelatin particles, etc., the sedimentation ring is clearly visible, and when the carrier particles are latex particles, the turbidity does not increase or is negligible.

Therefore, since the reaction between the antigen reagent and the antibody-sensitized particles is blocked in proportion to the amount of RF in the sample, a very small amount of RF in the sample can be measured, and the conventional modified Ig
The sensitivity is significantly higher than that of a reaction system in which RF in a sample is measured by G sensitized particles.

Further, IgG purified from serum of a patient with rheumatoid arthritis, denatured human or rabbit IgG, or a degradation fragment thereof, and a carrier particle sensitized with a synthetic antigen corresponding to an epitope recognized by RF are antigens. When it is used as a reagent, the agglutination reaction with antibody-sensitized particles occurs with higher sensitivity, and the amount of antigen-sensitized particles used can be further reduced, so that the detection sensitivity of RF in a sample is much higher. Become.

When the amount of RF in the sample is moderate, the reaction sites of the antigen reagent are reduced to about half, and the agglutination reaction with the antibody-sensitized particles occurs moderately. Therefore, when the carrier particles are red blood cells, gelatin particles, etc., the sedimentation ring expands, and when the carrier particles are latex particles, the increase in turbidity is about half.

In this reaction system, the specificity of the antibody sensitized to the carrier particles is directly linked to the accuracy of the determination or measurement result. In the case of a monoclonal antibody, the specificity can be easily determined at the cloning stage. Can be RF
It is easy to obtain high specificity for an antigenic determinant that specifically reacts with. In the case of a polyclonal antibody, a highly specific polyclonal antibody can be easily obtained by absorption with normal IgG, etc., and the presence of a plurality of non-specific reaction components for denatured IgG etc. is directly related to the specificity of the measurement result. It is much easier to secure the RF specificity as compared with the above method.

Further, as compared with the case where carrier particles are sensitized with an antigen such as denatured IgG, a reaction system with higher sensitivity can be obtained by sensitizing with antibodies, and the detection sensitivity can be further improved by using antigen-sensitized particles in combination. Is increased. Therefore, the required amount of the antigen reagent can be reduced, so that a smaller amount of the antibody can be detected and measured. When carrier particles are erythrocytes, gelatin particles, etc., highly specific semi-quantitative results can be obtained by a high dilution factor of the sample, and when carrier particles are latex particles, highly sensitive and highly specific quantification by aggregation inhibition rate. The statistical measurement result is obtained.

[0025]

【Example】

Example 1 Using human IgG isolated from serum of a patient with rheumatoid arthritis by ion exchange chromatography as an antigen,
Mice were immunized and monoclonal antibodies were prepared by a conventional method.

Mouse latex anti-RF monoclonal antibody was sensitized to commercially available latex particles having a particle size of 0.2 μm to give 0.1
% BSA-0.2M glycine-NaCl buffer (pH
The antibody-sensitized latex reagent was obtained by floating in 8.0).

The human IgG antigen was heated at 60 ° C. for 10 minutes, and this was added to 0.1% BSA-0.25M-2-amino-
It was diluted with 2-methyl-1,3-propanediol-HCl buffer (pH 8.0) to obtain an antigen reagent.

Sample serum 10 was added to 350 μl of the agglutinating antigen reagent.
μl is added and mixed, and after 5 minutes, 50 μl of antibody-sensitized latex reagent is added. The absorbance after 10 seconds and 300 seconds after addition of the antibody-sensitized latex reagent was measured at a wavelength of 570 nm,
The absorbance difference was determined.

The ratio of the absorbances blocked by the addition of the sample serum was calculated by setting the absorbance difference when the buffer solution was added instead of the sample serum as 100%.

The measurement results when a sample showing a blocking rate of more than 2SD of the average blocking rate of normal subjects is regarded as positive

[Table 1] It was shown to.

Example 2 Purified human IgG was treated at 60 ° C. for 10 minutes, digested with papain, and passed through a protein G column to obtain a bound fraction.
This was fractionated on a Sephacryl S300 column and human I
A gG.Fc fragment was obtained.

This was adsorbed and fixed on a flat bottom microtiter tray, washed and blocked with a BSA solution to prepare an antigen plate.

The mouse anti-RF monoclonal antibody was digested with pepsin to obtain (Fab ') 2, which was labeled with horseradish peroxidase (HRP) by a conventional method.

After washing the antigen tray, 100 μl of a 10-fold diluted test serum or standard solution was added to each well and the mixture was allowed to stand at room temperature for 2 hours.
Reacted for hours. After washing, 100 μl of the labeled antibody solution was added and reacted for 2 hours. After washing, a substrate solution (orthophenylenediamine / H 2 O 2) was added and reacted for 30 minutes.

After stopping the reaction with a 3N sulfuric acid solution, the absorbance was measured at 90 nm. Measurement results

As shown in FIG.

[0036]

As described above, according to the measuring method and the measuring reagent of the present invention, the following various excellent effects can be obtained.

1. Since the agglutination reaction between the antigen corresponding to RF and the antibody reagent is blocked by RF in the sample, the specificity of the reaction result becomes equal to the specificity of the antibody reagent, and the specificity of the antibody is either a monoclonal antibody or a polyclonal antibody. In this case also, it is easy to obtain a product with high specificity, and the specificity is higher than that in the case of measuring RF in a sample using a conventional modified IgG-sensitized particle.

2. When antibody-sensitized particles are used as the antibody reagent, the reaction with the antigen reagent occurs very sensitively, so that the amount of the antigen reagent used can be reduced and a small amount of R in the sample can be used.
F can be detected and measured, and the sensitivity is remarkably high as compared with the conventional reaction between modified IgG-sensitized particles and RF in a sample.

3. By using an antigen in which a solid layer is sensitized with an antigen that reacts with RF and a labeled antibody, a more sensitive measurement system can be obtained.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing an effect of the present invention.

Claims (18)

[Claims] 1. A method for measuring rheumatism factor, which comprises blocking an antigen-antibody reaction between an antigen corresponding to the rheumatism factor and an antibody reagent which specifically reacts with the antigen, by the rheumatism factor in the measurement sample. Method of measuring rheumatism factor. 2. The rheumatoid factor assay according to claim 1, wherein the index of the antigen-antibody reaction is an aggregation phenomenon of an antigen corresponding to the rheumatoid factor and carrier particles sensitized with an antibody that specifically reacts with the antigen. Method. 3. The method for measuring rheumatoid factor according to claim 2, wherein the carrier particles are red blood cells, synthetic resin particles, synthetic particles, liposomes, and metal colloids. 4. The rheumatism factor measurement method according to claim 2, wherein the reaction result is visually determined. 5. The method for measuring rheumatoid factor according to claim 2, wherein the reaction result is optically measured. 6. The method for measuring rheumatoid factor according to claim 2, wherein the antibody sensitized to the carrier particles uses RF isolated from RF-positive serum. 7. The method for measuring rheumatoid factor according to claim 2, wherein the antibody sensitized to the carrier particles is a monoclonal antibody. 8. The method for measuring rheumatoid factor according to claim 2, wherein the antibody sensitized to the carrier particles is a polyclonal antibody. 9. The method for measuring rheumatoid factor according to claim 2, which comprises sensitizing carrier particles with an antigen corresponding to the rheumatoid factor. 10. The method for measuring rheumatoid factor according to claim 9, wherein the carrier particles are red blood cells, synthetic resin particles, synthetic particles, liposomes, and metal colloids. 11. A rheumatoid factor assay reagent comprising an antigen corresponding to a rheumatoid factor and an antibody reagent that specifically reacts with the antigen. 12. The rheumatoid factor assay reagent according to claim 11, wherein the antibody reagent is carrier particles sensitized with an antibody that specifically reacts with an antigen corresponding to the rheumatoid factor. 13. The rheumatoid factor assay reagent according to claim 12, wherein the carrier particles are sensitized with an antigen corresponding to the rheumatoid factor. 14. The activity of a labeling substance such as a radioisotope, an enzyme or a chemiluminescent compound labeled with an antibody which specifically reacts with an antigen corresponding to a rheumatoid factor is used as an index of an antigen-antibody reaction. Method for measuring rheumatoid factors in. 15. A rheumatoid factor isolated from a rheumatoid factor-positive serum is used as an antibody reagent.
The method for measuring the rheumatoid factor described. 16. The method for measuring rheumatoid factor according to claim 14, wherein a monoclonal antibody is used as the antibody reagent. 17. The method for measuring rheumatoid factor according to claim 14, wherein a polyclonal antibody is used as the antibody reagent. 18. A rheumatoid factor comprising: a solid layer on which an antigen corresponding to the rheumatoid factor is immobilized; and an antibody reagent in which an antibody that specifically reacts with the antigen is labeled with an enzyme or a chemiluminescent compound. Measuring reagent.