JPS6363863B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoassays. More specifically, it relates to an immunoassay method that applies the binding properties of immunoglobulins.

It is well known that immunoglobulin antibodies bind to specific antigens or haptens, and are used in many immunoassays. Possible problems with such immunoassays include:
There is interference from other proteins contained in the body fluid being assayed. In particular, human serum contains rheumatoid factor (RA).
factor) and the complement component C ₁ q, which also bind to immunoglobulins. Furthermore, since the amounts of RA factor and C ₁ q in human serum differ from person to person, treatment to inactivate or remove endogenous RA factor and C ₁ q in serum is usually required before assay. Without this preprocessing, the test results will have significant errors.

In recent years, immunoglobulin F(ab') ₂ fragments have shown specificity for antigens and have been shown to be associated with RA factors or/and
Does not bind to C ₁ q (Fc fragment of immunoglobulin causes binding of immunoglobulin to RA factor or/and C ₁ q and does not bind to antigen)
Taking advantage of this property, RA factor or/and
As an immunoassay method that can be performed without interference even if C ₁ q is present, there is a method using an F(ab') ₂ fragment of immunoglobulin (Japanese Patent Application Laid-open No. 119292/1983). However, separating the F(ab') ₂ fragment of immunoglobulin from the Fc fragment requires complicated operations using column chromatography. In addition, it is generally considered that the influence of RA factor and/or C ₁ q cannot be avoided under the coexistence of F(ab') ₂ fragment and Fc fragment, and in fact, the coexistence of F(ab') ₂ fragment and Fc fragment The method described below, in which both antigen and antibody are reacted in a solution state to determine the amount of antigen present, is similar to the well-known method of assaying using whole immunoglobulin, in which the influence of RA factor and/or C ₁ q can be evaluated. receive.

As a result of extensive research, the present inventors have discovered that if latex particles are sensitized with a mixture of F(ab') ₂ and Fc fragments of immunoglobulin, RA factor and/or C. We have found that even if _1q exists, it is possible to perform an immunoassay that is free from interference. That is, the present invention provides an immunoassay method using immunoglobulin binding that can be performed without interference even if RA factor and/or C ₁ q is present in the sample. In particular, the method according to the present invention uses not only the F(ab') ₂ fragment among each fragment of immunoglobulin, but also
This is an immunoassay method characterized by using latex particles sensitized with a mixture of both F(ab') ₂ and Fc fragments of immunoglobulin.

Furthermore, the present invention provides the following methods: (a) A sample in a body fluid is treated with immunoglobulin F that exhibits specificity for a certain antigen (including hapten).
A mixture of the (ab′) ₂ fragment and the other fragment, the Fc fragment, is mixed with sensitized latex particles to create a reaction mixture.

(b) incubating the reaction mixture to react with the antigen present in the sample;

(c) Measuring the extent of the reaction and assaying the amount of antigen present in the body fluid sample.

This is an immunoassay method characterized by determining the amount of antigen present in a body fluid sample, which consists of steps (a), (b), and (c).

The mixture of immunoglobulin F(ab') ₂ fragment and Fc fragment used in the present invention is decomposed by a known method, for example, by enzymatic decomposition using pepsin, and the decomposed mixture is used as it is without being decomposed. An example of preparing a decomposition mixture is shown below.

Preparation example: Dissolve 100 mg of immunoglobulin in distilled water to make 10 ml, and add crystalline citric acid to adjust the pH to 4.5. Next, after adding 0.2 mg of pepsin, the cells were incubated at 37°C for 96 hours to form immunoglobulin F(ab') ₂ fragments.
Obtain a mixture of Fc fragments.

The present invention provides for the use of whole immunoglobulins or immunoglobulins in immunoassays that generate a specific reaction between an antigen and an immunoglobulin, but do not require a reaction between factor RA or/and C ₁ q and the immunoglobulin. Immunoglobulin F(ab') ₂ instead of sensitized latex particles with globulin F(ab') ₂ fragment alone
Mixture sensitized latex particles of fragments and Fc fragments can be widely used.

The above-mentioned specific binding between the mixture sensitized latex particles and the antigen causes aggregation of the latex particles. The degree of aggregation can be qualitatively determined by observing with the naked eye, and quantitatively by automatically counting unaggregated particles or by irradiating the agglomerated particles with light and measuring the absorbance.

Furthermore, the immunoglobulin F (ab') ₂ fragment of the present invention and Fc
The mixture of fragments sensitized latex particles can be used as an immunochemical separation reagent for the immunochemical separation of trace antigenic substances. That is, it is used as an immunochemical separation reagent for a second antibody used in radioimmunoassay or enzyme antibody method.

The quantitative assay method using the immunoassay method according to the present invention is preferably a continuous flow method, and the semi-quantitative assay method can be carried out by a rapid measurement method that is visually determined on a reaction plate or slide plate.

EXAMPLES The present invention will be explained in detail with reference to Examples below.

Example 1 Preparation of F(ab') ₂ fragment and Fc fragment mixture sensitized latex particles by physical binding.
% suspension is diluted 30 times with 0.2M ammonium/0.154M sodium chloride buffer, pH 8.2, and centrifuged twice with this buffer. 10 mg F(ab') ₂ per ml
The fragment, Fc fragment mixture solution, and latex suspension prepared at a concentration of 2% with the above buffer were mixed at a ratio of 1:20, and incubated at 37°C for 120 minutes.
The excess F(ab') ₂ fragment and Fc fragment mixture was centrifugally washed using the above buffer. Next, add 1% bovine serum albumin to the buffer solution to 1.0%, that is, compared to the initial concentration.
Make a latex suspension that is diluted 30 times.

Preparation of antiserum CRP antiserum was prepared by immunizing a rabbit with Freund's complete adjuvant.

Automatically Operated Assay Patient serum is absorbed at a rate of 0.1 ml per second into a continuous flow system consisting of a constant flow pump, manifold, particle counter and recorder. The serum was mixed with 0.5 ml of latex particles suspended in ammonium buffer;
Pass through the incubating coil for 20 minutes. This solution is then passed through a particle counter, where unaggregated particles are counted and CRP in the serum is measured. CRP concentration in serum could be measured in the range of 1 ng/ml to 100 ng/ml.

Furthermore, as a result of measurements using serum samples from 10 patients, a CV value of 5% was obtained, and the correlation coefficient when compared with the product obtained by one-dimensional immunodiffusion method using similar samples was 0.93. Indicated.

Example 2 Preparation of latex particles sensitized with F(ab') ₂ fragment and Fc fragment mixture by chemical bonding A 10% suspension of carboxylated latex particles (manufactured by Dow, diameter 0.255μ) was mixed with 0.1M tris-hydrochloric acid/
Dilute 10 times with 0.154M sodium chloride buffer (PH8.4). A 1% concentration F(ab') ₂ fragment Fc fragment mixed solution is mixed therewith at a ratio of 40:1 to cause covalent bonding. After centrifugal washing in the same manner as in Example 1, a 1% suspension is prepared with 0.1M Tris-HCl/0.154M buffer (PH8.4) containing human serum albumin.

Preparation of antiserum HPL antiserum was prepared by immunizing a goat using Freund's complete adjuvant.

Manually operated assay: 0.1 ml of the F(ab') ₂ fragment prepared above and the Fc fragment mixture sensitized latex reagent and 0.1 ml of the above Tris buffer.
and are mixed in an acrylic resin cell (layer length 10 mm).

Next, add 0.1 ml of pregnant human serum and incubate at 37°C for 30 minutes while shaking and mixing. After that, add 3.1ml of the above buffer solution, shake well, and then
Measure the absorbance of
Measure HPL concentration.

HPL concentration in serum is 2ng/ml to 500ng/ml
It was possible to measure within the range of . The correlation coefficient with the results obtained by radioimmunoassay method was 0.95. The results in 20 male RA patient serum cases were
The HPL concentration was 5 ng/ml or less, indicating that it was not affected by the RA factor.

Claims (1)

[Claims] 1. A mixture of immunoglobulin F (ab') ₂ fragment and Fc fragment is sensitized to latex particles having an average particle size of 0.1 μ to 1.0 μ, and the obtained sensitized latex particles and body fluid An immunoassay method that consists of reacting with an antigen in the body. 2. Convert body fluid samples into immunoglobulin F (ab') ₂ fragments and Fc
A mixture with fragments is mixed with sensitized latex particles to form a reaction mixture, and the degree of latex agglutination caused by the reaction between the antigen in the sample and the immunoglobulin F(ab') ₂ fragment that exhibits specificity for the antigen is visually observed. or by irradiating light with a wavelength of 0.3μ to 1.8μ,
2. The immunoassay method according to claim 1, wherein the amount of the antigen present is determined by measuring its absorbance or intensity of scattered light. 3. Claims 1 or 2, characterized in that the degree of latex aggregation is determined by selectively counting unaggregated latex particles or aggregated latex particles remaining in the reaction mixture to determine the amount of antigen present. immunoassay.