JP2541826B2 - Immunoassay - Google Patents

Description

TECHNICAL FIELD The present invention relates to an optical immunoassay method using an antigen-antibody reaction for quantifying an antigen in a specimen.

[Prior Art] Various immunoassays for measuring an antigen in a sample, such as a single radial immunodiffusion method, a laser nephelometric method, a nephelometric method, a radioimmunoassay method, an enzyme immunoassay method, and a latex agglutination method, are available. It has been put to practical use. Recently, among these, especially the laser ratio method, which is quick and sensitive, and easy to automate,
Optical immunoassays such as nephelometry have become widely used. In these optical immunoassays, a sample containing an antigen to be measured and an antibody solution containing an antibody corresponding to the antigen are mixed to produce an insoluble antigen-antibody complex, and the absorbance and scattering at that time Measure the change in light intensity. Since the amount of the antigen-antibody complex is increased as the amount of the antigen in the sample increases, the amount of the antigen in the sample can be measured by the magnitude of the optical change amount. The measurement of the amount of antigen is automatically performed within a short time using a laser nephelometer or an automatic analyzer developed for biochemical measurement of multiple items.

[Disadvantages of Prior Art] Conventionally, when the antigen contained in a specimen is quantified by irradiating the antigen-antibody complex generated by the antigen-antibody reaction with light to determine the optical change amount thereof, Due to the quantitative ratio, it is difficult to obtain a direct proportional relationship between the antigen concentration and the amount of optical change. In particular, when the antigen concentration becomes high, the optical change amount rapidly decreases, and when a calibration curve is created, there is one corresponding antigen concentration in each of the low concentration region and the high concentration region for one optical variation amount. In some cases, immunoquantification based on the amount of optical change becomes impossible in this case. Furthermore, when immunoassaying the antigen in the sample,
Although there is a demand to measure the sample as it is without diluting it, the above problem becomes more serious when the sample is measured as it is because the amount of antigen increases.

[Problems to be Solved by the Invention] Accordingly, an object of the present invention is to provide a direct proportional relationship between the amount of antigen in a sample and the amount of optical change, regardless of the ratio of the amount of antigen in the sample and the antibody to be acted on. The aim is to provide an optical immunoassay in which the relationship always holds.

[Means for Solving Problems] As a result of earnest research, the present inventors
Fab fragment or F of antibody corresponding to the antigen to be measured
When it is carried out in the presence of the ab 'fragment, the Fab fragment or the Fab' fragment competes with the antibody for an antigen-antibody reaction, and a clean direct proportional relationship can be established between the antigen concentration and the optical change amount. And completed the present invention.

That is, the present invention, the antigen to be measured in the sample,
In an immunoassay in which an antigen-antibody complex is produced by reacting with an antibody corresponding to the antigen and the produced antigen-antibody complex is irradiated with light to measure an optical change, The immunoassay method is characterized in that the reaction is carried out in the presence of a Fab fragment or Fab 'fragment of an antibody corresponding to the antigen.

[Effects of the Invention] According to the immunoassay method of the present invention, there is a direct proportional relationship between the amount of antigen to be measured in a sample and the amount of optical change, irrespective of the amount ratio of the antigen and the antibody. Therefore, even when the antigen to be measured is contained in a high concentration in the sample, the optical immunoassay method can be performed as a stock solution without diluting the sample. Therefore, the time-consuming and labor-intensive work of accurately diluting the sample can be omitted, and further, the application to an automatic analyzer for carrying out a positive chemical test for the same sample becomes extremely easy, which is very advantageous. .
Further, since the antigen concentration and the amount of optical change are in direct proportion, measurement can be performed with only one concentration of standard solution.

[Detailed Description of the Invention] As described above, in the method of the present invention, the antigen-antibody reaction between the antigen to be measured and the antibody in the sample is determined by the Fab fragment or Fab 'fragment of the corresponding antibody of the antigen to be measured. In the presence of. The Fab fragment is a fragment from the hinge region of the immunoglobulin to the N-terminus and can be obtained by papain-degrading immunoglobulin. Similarly, the Fab ′ fragment is a fragment from the hinge region of an immunoglobulin to the N-terminal, and is F (a) obtained by pepsin degradation of an immunoglobulin.
b ') obtained by reducing ₂ fragments. Methods for preparing Fab fragments and Fab ′ fragments are widely known, and for example, RRParter, Biochem.J., 7
3 , 119 (1959). Fab fragment and
The Fab 'fragment does not substantially cause aggregation even when it binds to an antigen to form an antigen-antibody complex, and thus does not change the amount of optical change.

The Fab fragment or Fab 'fragment used is derived from an antibody corresponding to the antigen to be measured.
The antibody from which these fragments originate may be any antibody as long as it corresponds to the antigen to be measured, and it may be a monoclonal antibody or a polyclonal antibody. Further, the animal species serving as a source of the antibody is not limited, and may be the same species or a different species from the animal species that supplies the antigen used to form the antigen-antibody complex at the time of immunoassay. The fragment may be derived from a single antibody or a mixture derived from a plurality of different antibodies.

The concentration of Fab fragment or Fab ′ fragment is not particularly limited, but the Becker titer is preferably about 0.3 mg / ml to 6.0 mg / ml in the reaction mixture. The Becker titer is the amount of antigenic protein (mg) that reacts with 1 ml of antiserum (Reference: W. Becker. Immunochemistr.
y, 6: 539-546 (1969)).

In the optical immunoassay method of the present invention, the antigen to be measured in the sample is reacted with the antibody in the presence of the Fab fragment or Fab 'fragment described above, that is, in the competition between these fragments and the antibody. The sample that can be used in the method of the present invention may be any sample as long as it may contain an antigen, and examples thereof include body fluids such as serum, urine and saliva.
These body fluids can be used for measurement without being diluted. The antigen to be quantified may be any antigen as long as it has antigenicity, and examples thereof include immunoglobulin, complement, transferrin, haptoglobin, and C-reactive protein.

The antibody reacted with the antigen to be measured in the sample to form an antigen-antibody complex that causes aggregation may be any antibody as long as it corresponds to the antigen to be measured, and it may be a monoclonal antibody. It may be a polyclonal antibody or an antiserum containing the polyclonal antibody. In addition, the animal species that is the source of the antibody is not limited.
Also, a single antibody can be used, or a plurality of antibodies can be mixed and used. The concentration of the antibody is usually 2 mg / ml to 20 mg / ml in the reaction mixture.

The antigen-antibody reaction may be carried out under any conditions as long as the reaction occurs, and is usually at 30 ° C to 37 ° C for about 2 minutes to 10 minutes.

After the initiation of the antigen-antibody reaction, the reaction mixture is irradiated with light as in the conventional case, and the optical change amount thereof is measured. The optical change amount is the change amount of absorbance or the change amount of scattered light, and these can be automatically measured by a commercially available automatic analyzer. When the amount of antigen in a sample is large, a large amount of antigen-antibody complex is formed and aggregation also occurs strongly. Conversely, when the amount of antigen is small, aggregation is weak. You can know the amount of antigen that should be used.

[Examples of the Invention] Reference Example Fab fragments were prepared from anti-human IgG serum by the method described in RRParter, Biochem.J., 73 , 119 (1959). That is, 1 ml of 100 mM L-cysteine-hydrochloride, 20 mM EDTA.2Na solution (pH 7.0) and 0.1 mg of papain were added to 10 ml of anti-human IgG serum to adjust the pH to 5-6. After incubating at 37 ° C. for 3 hours, iodoacetamide was added to 10 mM, and the mixture was further incubated at 37 ° C. for 30 minutes to inactivate papain. Then, it was dialyzed against 0.1 M Tris buffer (pH 8.0). The insoluble matter in the antiserum was separated by centrifugation and further filtered through a 0.45 μm membrane filter.

The Fab fragment of the antiserum thus obtained and the untreated anti-human IgG serum were each treated with 0.1 M glycine buffer (p
It was diluted 4-fold with H8.0), a human IgG 3000 mg / dl-containing solution was added, and the change in absorbance per minute was measured at a wavelength of 340 nm using a biochemical automatic analyzer TBA-480 (manufactured by Toshiba). Further, as a blank, the same measurement was performed using purified water instead of the human IgG-containing solution.

The results are shown in Fig. 1. In FIG. 1, A shows the result for the mixture of the Fab fragment and the human IgG-containing solution, a shows the result for the mixture of the Fab fragment and purified water, and B shows the untreated anti-antibody. Human
The results for a mixture of IgG serum and a human IgG-containing solution are shown, and b shows the results for a mixture of anti-human IgG serum and purified water. As is clear from FIG. 1, the untreated antiserum and the human IgG undergo an antigen-antibody reaction and strongly aggregate, and the untreated antiserum contains the anti-human IgG antibody. Further, when the untreated antiserum is mixed with purified water, no aggregation is observed at all, and therefore the untreated antiserum itself does not cause aggregation. On the other hand, no aggregation is observed when the Fab fragment derived from the untreated antiserum is mixed with the human IgG antibody-containing solution or purified water. From the above, it can be seen that the Fab fragment does not aggregate at all even when an antigen-antibody reaction is caused to form an antigen-antibody complex.

Example 1 5 μl of a physiological saline solution of human IgG having a concentration of 0 to 3000 mg / dl
After adding 300 μ of 20 mM glycine buffer (pH 9.5) to
An antibody solution (300 μm) containing the Fab fragment (1.1 mg (Becker titer)) obtained in the above Reference Example and the antibody (2.9 mg, Becker titer) was added. As a control, 300 μ of antibody solution containing no Fab fragment (antibody amount 1.1 mg (Becker titer) and 2.9 mg (Becker titer)) was added, and the change in absorbance per minute using the automatic analyzer used in the reference example. Was measured at a wavelength of 340 nm.

Results are shown in FIG. In FIG. 2, the black circles show the results when the antigen-antibody reaction was carried out in the presence of Fab by the method of the present invention, and the white circles and triangles show the antibody amounts of 1.1 mg and 2.9 mg in the absence of Fab, respectively. The results are shown below. As is clear from FIG. 2, in the case of only the antibody, when the concentration of human IgG was high, regardless of the amount of antibody,
The aggregation reaction becomes faster and the amount of change in absorbance per minute becomes smaller. However, when the Fab fragment and the antibody are mixed according to the method of the present invention, a direct proportional relationship is observed between the IgG concentration and the amount of change in absorbance per minute.

Example 2 This example is intended to show that the presence of Fab fragments does not compromise measurement accuracy and accuracy.

Using the IgG standard sample of known concentration, the same operation as in Example 1 was performed to prepare a calibration curve. Next, for various unknown concentrations
The same operation as in Example 1 was performed using an undiluted sample containing IgG as a raw solution, and from the measurement results and the above calibration curve,
The IgG concentration was determined.

On the other hand, the concentration of the same sample was measured based on a conventional method known as the one-way radioimmunodiffusion method, which has been widely known, and compared with the result obtained by the method of the present invention.

A correlation diagram of the measured values by the above-mentioned two methods is shown in FIG. The correlation coefficient of this correlation diagram is 0.951, and it can be seen that the measured values by the above two methods are almost the same. That is, in the method of the present invention, it was revealed that the presence of Fab fragments does not adversely affect the measurement accuracy and accuracy.

[Brief description of drawings]

FIG. 1 is a diagram showing the time course of the absorbance of the Fab fragment obtained in the reference example and the untreated antiserum, and FIG. 2 is a diagram showing the relationship between the human IgG and the rate of absorbance change obtained in Example 1. FIG. 3 is a correlation diagram between the measured value by the method of the present invention and the measured value by the one-way radiation immunodiffusion method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shosaku Motoda 1-2-2 Minamihonmachi, Gosen-shi, Niigata Denka Seiken Co., Ltd. Niigata Plant

Claims (4)

(57) [Claims] 1. An antigen-antibody complex is produced by reacting an antigen to be measured in a specimen with an antibody corresponding to the antigen, and the produced antigen-antibody complex is irradiated with light to obtain an optical change amount. In the immunoassay method for measuring the antigen, the reaction between the antigen and the antibody is carried out by changing the Fab fragment or Fab ′ of the antibody corresponding to the antigen.
An immunoassay method characterized by being performed in the presence of a fragment. 2. The immunoassay method according to claim 1, wherein the concentration of the Fab fragment or Fab ′ fragment is 0.3 to 6.0 mg / ml in Becker titer. 3. The immunoassay method according to claim 1 or 2, wherein the optical change amount is a change amount of the light scattering intensity. 4. The immunoassay method according to claim 1 or 2, wherein the optical change amount is a change amount of the transmitted light amount.