JP2678273B2 - 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 methods, optical immunoassay methods such as laser nephelometry and nephelometry have come into widespread use because of their rapid measurement, high sensitivity, and easy automation. In these optical immunoassays, an insoluble antigen-antibody complex is produced by mixing a sample containing an antigen to be measured with an antibody solution containing an antibody corresponding to the antigen,
At that time, changes in absorbance and scattered light intensity are measured. 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] In actual diagnosis, the amount of antigen to be measured in a sample is unknown before measurement, and when processing a large number of samples, the concentration and amount of the antibody solution added to each sample are the same. Is.
The concentration and amount of the antibody solution are selected so that the amount of antigen can be accurately measured even when the amount of antigen in the sample is large. Therefore,
In a sample with a small amount of antigen, the amount of antibody becomes excessive and it becomes difficult to form an antigen-antibody complex, and as a result, there is a drawback that the antigen cannot be quantified accurately. Further, when immunoassaying an antigen in a sample, there is a demand to measure the sample as it is without diluting the sample, but when measuring as it is, a large amount of antibody acts. Therefore, the problem of antibody excess described above becomes even more serious. [Problems to be Solved by the Invention] Accordingly, an object of the present invention is to provide an optical immunoassay for accurately quantifying an antigen even when the antigen to be measured is contained in a sample at a low concentration. To provide the law. [Means for Solving Problems] As a result of earnest research, the inventors of the present invention have made an antibody corresponding to an antigen to be measured act on an analyte in the conventional optical immunoassay method. By forming an antigen-antibody complex in advance, it was found that the antigen can be accurately quantified even when the sample to be measured contains a low concentration of the antigen, and the present invention was completed. That is, the present invention, the antigen to be measured in the sample,
An antigen-antibody complex formed by reacting a first antibody corresponding to the antigen to produce an antigen-antibody complex, and then reacting the antigen-antibody complex with a second antibody corresponding to the antigen An immunoassay method is provided, which comprises irradiating the skin with light and measuring the amount of optical change. [Effects of the Invention] According to the method of the present invention, even when the concentration of the antigen to be measured in a sample is wide ranging from low to high, the same concentration and amount of each sample can be obtained. The amount of antigen in each sample can be accurately measured using the antibody solution. DETAILED DESCRIPTION OF THE INVENTION In the present invention, first, a sample containing an antigen to be measured is
The first antibody against the antigen to be measured is allowed to act to form an antigen-antibody complex. The first antibody may be any as long as it corresponds to the antigen to be measured,
It may be a monoclonal antibody or a polyclonal antibody.
Further, the animal species serving as the source of the antibody is not limited, and it may be the same animal as the second antibody described below or a different animal. In addition, as the first antibody, a single antibody can be used, or a plurality of antibodies can be mixed and used. The amount of the first antibody added to the sample is such that aggregation does not substantially occur due to the formation of the antigen-antibody complex. Further, when the corresponding antigenic determinant is the same as the second antibody described later, the amount is such that the free antigenic determinant remains after the reaction with the first antibody. That is, the first antibody is usually mixed with the analyte in the form of a solution, in which case the first antibody
The concentration of the antibody solution is not particularly limited, but is usually about 5 mg / ml to 30 mg / ml. In addition, the mixing ratio of the first antibody solution and the sample is the amount of the second antibody, whether the first antibody is a polyclonal antibody or a monoclonal antibody, and
The content of the first antibody solution in the mixed solution of the first antibody solution and the specimen is usually 0.01 v / v% to 5.0 v / v, although it depends on whether the antibody and the second antibody are derived from the same animal or different animals. %. However, the mixing ratio of the first antibody solution is large when the amount of the second antibody is large, it is large when the first antibody is a monoclonal antibody, and the origin of the first antibody and the second antibody is the same species. However, it is not necessarily limited to the above range. The reaction condition between the first antibody and the sample may be any condition as long as an antigen-antibody reaction occurs, and usually 30 ° C to 37 ° C.
It is about 1 to 10 minutes at ℃. Further, the sample that can be used in the method of the present invention,
Any substance may be used 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, C-reactive protein, α-fetoprotein, haptoglobin and transferrin. Next, the second antibody is added to the mixture of the sample and the first antibody. The second antibody may be any antibody as long as it corresponds to the antigen to be measured, and may be a monoclonal antibody or a polyclonal antibody. The antigenic determinant of the second antibody may be the same as or different from the antigenic determinant of the first antibody. Therefore, the second antibody may be the same antibody as the first antibody. Further, the animal species serving as the source of the antibody is not limited, and it may be an animal of the same species as the first antibody or an animal of a different species. The second antibody may be a single antibody or a mixture of a plurality of antibodies. The second antibody is usually mixed with the analyte in the form of a solution.
In this case, the concentration of the first antibody solution is not particularly limited,
Usually, it is about 5 mg / ml to 30 mg / ml. Further, the mixing ratio of the first antibody solution and the sample varies depending on the amount of the first antibody, whether the second antibody is a polyclonal antibody or a monoclonal antibody, and whether the first antibody and the second antibody are derived from the same animal or different animals. However, the content of the first antibody solution is usually 0.01 v / v% to 5 v / v% in the mixed solution of the first antibody solution and the specimen. The mixing ratio of the first antibody solution is large when the amount of the second antibody is large, is small when the second antibody is a monoclonal antibody, and is small when the animal from which the first antibody and the second antibody are derived is the same species. However, the number is not necessarily limited to the above range. The reaction condition between the second antibody and the sample may be any condition as long as an antigen-antibody reaction occurs, and it is usually 30 ° C to 37 ° C.
It is about 1 to 10 minutes at ℃. Immediately after adding the second antibody, the reaction system is irradiated with light,
The amount of optical change is measured. The optical change amount is the change amount of the transmitted light amount (that is, the change amount of the absorbance) or the change amount of the scattered light intensity. The measurement of the amount of optical change can be automatically performed using a commercially available automatic analyzer conventionally used in this field. When the amount of the antigen in the sample is large, a large amount of the antigen-antibody complex is generated and aggregation rapidly occurs, so that the optical change amount becomes large. Therefore, if a calibration curve is prepared based on a sample of known concentration, the antigen in the sample of unknown concentration can be quantified. [Examples of the Invention] Example 1 5 μl of a physiological saline solution of human IgG having a concentration of 0 to 3200 mg / dl.
In addition, as the first antibody, a solution of anti-human IgG monoclonal antibody with a concentration of 12.8 mg / ml (antibody titer 10,000 times by ELISA method) ^{* 1}
Was added to 300 μl of a 20 mM glycine buffer solution (pH 9.5) containing 1.0 v / v% of the mixture, and the mixture was reacted at 37 ° C. for 5 minutes. On the other hand, as a control, 300 μl of the same buffer solution containing no anti-human IgG monoclonal antibody as the first antibody was added to the above human IgG solution, and the same reaction was performed. 300 μl of anti-human IgG serum was added to each mixture as the second antibody, and the change in the absorbance during 1 to 3 minutes after the reaction was started using an automatic analyzer (Toshiba TBA-480) while reacting at 37 ° C. The measurement was carried out at a wavelength of 340 nm, and the rate of change in absorbance per minute was calculated from the measurement results. The results are shown in FIG. In FIG. 1, the solid line shows the result when both the first antibody and the second antibody were acted according to the method of the present invention, and the broken line is the result when only the second antibody was acted according to the conventional method. Show. As is apparent from FIG. 1, when the first antibody was not added, the absorbance change rate proportional to the IgG concentration could not be obtained when the IgG concentration was 1600 mg / dl or less due to excess antibody, but when the monoclonal antibody was added. A change rate in absorbance proportional to the IgG concentration was obtained. Thus, according to the method of the present invention,
It can be seen that it is difficult to measure by the conventional immunoturbidimetric method and other immunoassay methods, and it becomes possible to accurately quantify the antigen from low concentration to high concentration. * 1 A monoclonal antibody obtained by proliferating a hybridoma obtained by fusing BALB / c mouse spleen cells immunized with human IgG and mouse myeloma cell P3U1 by a conventional method in the abdominal cavity of BALB / c mice. Example 2 5 μl of a physiological saline solution of human IgG having a concentration of 0 to 3200 mg / dl
In addition, a 20 mM glycine buffer solution (pH9.10) containing 0.4 v / v% of an anti-human IgG polyclonal antibody solution having a concentration of 15 mg / ml as the first antibody.
5) 300 μl was added and reacted at 37 ° C. for 5 minutes. On the other hand, as a control, anti-human I as the first antibody was added to the above human IgG solution.
300 μl of the same buffer containing no gG polyclonal antibody was added and reacted in the same manner. As the second antibody, 300 μl of anti-human IgG serum containing the same antibody as the above-mentioned first antibody was added to each mixture, and the reaction was carried out at 37 ° C. while using an automatic analyzer (Toshiba TBA-480). The change in absorbance between minutes and 3 minutes was measured at a wavelength of 340 nm, and the rate of change in absorbance per minute was calculated from the measurement results. The results are shown in FIG. In FIG. 2, the solid line shows the result when both the first antibody and the second antibody were acted according to the method of the present invention, and the broken line is the result when only the second antibody was acted according to the conventional method. Show. As is clear from FIG. 2, as in Example 1, anti-human IgG
When the polyclonal antibody was added, the absorbance change rate proportional to the IgG concentration was obtained. Further, in the method of the present invention, it was found that not only the monoclonal antibody used in Example 1 but also a polyclonal antibody can be used as the first antibody to perform accurate immunoassay. Example 3 This example is intended to show that the presence of the first antibody does not reduce the measurement accuracy and precision. Using the IgG standard sample of known concentration, the same operation as in Example 2 was performed to prepare a calibration curve. Next, for various unknown concentrations
The same operation as in Example 1 and Example 2 was performed using a sample containing the IgG antibody as it was, and the IgG concentration in the sample was determined from the measurement results and the calibration curve. On the other hand, the concentration of the same sample was measured based on the conventional one-way radiation immunodiffusion method which has been widely known, and the result was 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 was 0.958, and it was found that the measured values by the above-mentioned two methods were almost the same. That is, it was revealed that the presence of the first antibody did not adversely affect the measurement accuracy and precision in the method of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a calibration curve when a monoclonal antibody is used as the first antibody and a calibration curve by a conventional method, and FIG. 2 shows when a polyclonal antibody and the first antibody are used. FIG. 3 is a diagram showing a calibration curve and a calibration curve by a conventional method, and FIG. 3 is a correlation diagram of the result of immunoassay by the method of the present invention and the result of immunoassay by the one-way radiation immunodiffusion method.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Shuichi Meguro               1-2-2 Minamihonmachi, Gosen City, Niigata Prefecture               Nka Seiken Co., Ltd. Niigata Factory (72) Inventor Mori Sekine               1-2-2 Minamihonmachi, Gosen City, Niigata Prefecture               Nka Seiken Co., Ltd. Niigata Factory (72) Inventor Shosaku Motoda               1-2-2 Minamihonmachi, Gosen City, Niigata Prefecture               Nka Seiken Co., Ltd. Niigata Factory                (56) References JP-A-60-237363 (JP, A)

Claims (1)

(57) [Claims] The antigen to be measured in the sample and the first corresponding to the antigen
To produce an antigen-antibody complex, and thereafter, the antigen-antibody complex is reacted with a second antibody corresponding to the antigen, and the formed antigen-antibody complex is irradiated with light to generate an optical spectrum. Immunoassay consisting of measuring the dynamic change. 2. The immunoassay method according to claim 1, wherein the optical change amount is a change amount of the light scattering intensity. 3. The immunoassay method according to claim 1, wherein the amount of optical change is the amount of change in transmitted light.