JP2003004741A - Method for judging antigen-antibody reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a judgment method which can establish an immunological measurement method whereby correct measurement results can be obtained irrespective of whether or not a nonspecific agglutination reaction takes place. SOLUTION: In the method for judging an antigen-antibody reaction to measure an antibody or antigen corresponding to a carried antigen or antibody with the use of an insoluble carrier which carries the antigen or antibody, for a reagent constitution for the measurement, reagents of two or more liquids comprising a suspension of the insoluble carrier and a buffer solution are used, and these reagents are mixed at a measurement time. The measurement is carried out twice. For a specimen which brings about a reaction at a first measurement time, a second measurement is carried out after replacing the buffer solution provided for the measurement in the reagent constitution with a buffer solution which includes the same antibody or antigen as carried to the insoluble carrier. Whether it is a specific reaction or a nonspecific reaction is judged by measuring a decrease degree of the reaction with the buffer solution to the first reaction.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention belongs to the field of immunological measurement, and more particularly to a measurement utilizing an antigen-antibody reaction, and the measurement results are simple and highly accurate. It relates to a judgment method. [0002] An antigen-antibody reaction is routinely used for detecting a trace component. In an immunological measurement method, it is important to determine whether the measured result is due to an antigen-antibody reaction. For example, JP
No. 2-80956 discloses that on a substrate surface such as a slide, an agglutination reagent is mixed, an agglutination image resulting from an antigen-antibody reaction is observed and determined, and then the pH is adjusted to 3.0 or less. Alternatively, the concentration is changed to 9.0 or more, and the ion concentration is set to 0.5M or more, and a chemical substance containing a high concentration of chaotropic ions is added to determine whether or not aggregation is dissociated.
A method for confirming non-specific aggregation is disclosed. [0003] Also, Japanese Patent Application Laid-Open No. 11-511255 discloses that a reagent containing an immobilizing material in which a specific binding member specific to an IgM antibody is immobilized, bound to a solid phase material, and then converted from an IgM antibody to an IgG antibody. A method is disclosed in which the pH is determined to be sufficiently low to cause a divergence. [0004] A method using an immune reaction is a method for specific antigen-
Since the antibody reaction is used, the method has high measurement specificity. However, a non-specific agglutination reaction may occur depending on the state of the sample, and an error may occur in the measurement result. [0005] In order to solve such a problem, as a means for confirming whether an antigen-antibody reaction or a non-specific agglutination reaction occurs, in the above-described example, the deviation of the antigen-antibody reaction when the reaction occurred is an antigen-antibody reaction. Judgment is made based on conditions (change in pH, ionic strength, change in chaotropic ion, etc.), but in such a method, dissociation may occur even in non-specific reactions other than antigen-antibody reaction, It may not be possible to determine whether it is an antigen-antibody reaction. [0006] In view of the above situation, the present invention provides an immunological measurement method capable of obtaining an accurate measurement result regardless of whether a non-specific agglutination reaction has occurred. It is an object to provide a determination method that can be established. [0007] The present invention provides a method for determining an antigen-antibody reaction in which an insoluble carrier carrying an antigen or an antibody is used to measure an antibody or an antigen corresponding to the carried antigen or the antibody. The reagent configuration for the measurement uses two or more reagents consisting of a suspension of an insoluble carrier and a buffer, and these reagents are mixed at the time of measurement.
The measurement is performed twice, and for the sample that has undergone a reaction at the time of the first sample measurement, the buffer to be used for measurement in the reagent composition contains the same antibody or antigen as that supported on the insoluble carrier. After replacing the buffer, the second measurement is performed, and the degree of decrease in the reaction with the buffer relative to the first reaction is measured to determine whether the reaction is a specific reaction or a non-specific reaction. This is a method for determining an antigen-antibody reaction. Hereinafter, the present invention will be described in detail. In the present invention, a suspension of an insoluble carrier,
Replace the buffer consisting of a normal measurement reagent consisting of a buffer with a buffer containing an antibody or antigen having the same specificity as the antibody or antigen previously carried on an insoluble carrier (hereinafter referred to as "neutralization buffer"). The measurement is performed again while consuming the antigen or antibody in the sample. By taking such a measure, it is possible to check whether the antigen / antibody has been consumed without having to transfer the sample to another container, and it is very easy to carry out the antigen-antibody reaction while maintaining the specificity. Confirmation can be performed. As antigens adsorbed or bound to an insoluble carrier and contained in the neutralization buffer, protein antigens, lipid antigens, haptens, and haptens include carriers (eg, BSA).
The properties thereof are not limited as long as they can be used in immunoassays, such as those bound to proteins such as Also,
In the case of a purified protein or the like, animal species, tissues and the like need not be of the same origin as long as they have the same specificity as those adsorbed or bound to an insoluble carrier. As the antigen to be adsorbed or bound to the insoluble carrier and contained in the neutralization buffer, either a polyclonal antibody or a monoclonal antibody can be used, and an enzyme-digested fragment thereof is used. You can also. Further, the antibodies adsorbing and binding to the insoluble carrier need not necessarily be antibodies of the same species, and one may be a monoclonal antibody and the other may be a polyclonal antibody. The measuring system is not limited as long as it is composed of a latex suspension, a buffer or the like. That is, a reagent configuration in which the buffer solution is divided into two or more types and the latex suspension is divided into two or more types may be used. If at least two types of latex suspension and one type of buffer are used at the time of measurement, two or more types of buffer and two or more types of latex suspension are stored during storage. The method does not hinder the present invention. The neutralization rate is not limited to a numerical value. In order to facilitate the determination, it is preferable that the neutralization ratio can be set to a high value of 50% or more as a criterion. However, even when it is difficult to obtain high titer antibodies, highly purified antigens, etc. The present invention is not hampered as long as the presence or absence can be determined. The present invention will be described below in more detail by giving Examples and Comparative Examples of the present invention. The present invention is not limited to the following examples. (Example 1) Application to HBs antigen measurement reagent-1 1) Preparation of first reagent (buffer) (1) Preparation of first reagent for normal measurement 1% by weight of bovine serum albumin (hereinafter also referred to as BSA) Phosphate-salt buffer solution (0.05 M phosphate buffer (pH 7.0), 0.1 M NaCl;
Polyethylene glycol having an average molecular weight of 500,000 (manufactured by Wako Pure Chemical Industries, Ltd .; hereinafter, also referred to as PEG) was dissolved at a concentration of 1.0% by weight in 05 MPBS.
To 1 L of the above solution, 3.0 μl of normal rabbit serum was further added.
L was added to prepare a first reagent for normal measurement. (2) Preparation of first reagent for neutralization PEG was added to 0.05M PBS containing 1% by weight of BSA.
Was dissolved to a concentration of 1.0% by weight. To 1 L of the above solution, 3.0 μL of an anti-HBs antiserum (rabbit) (specific antibody titer: 8000 IU / mL (measured with a media Ace HBs antibody (manufactured by Sekisui Chemical Co., Ltd.)) was added.
A first reagent for neutralization was prepared. 2) Preparation of second reagent (anti-HBs antibody-sensitized latex solution) Phosphate-salt buffer (0.036 M phosphate) containing anti-HBs rabbit antibody (purified product) at a concentration of 1.0 mg / mL Buffer solution (pH 6.6), 0.1 M NaCl;
036 MPBS) in 1.25 mL of polystyrene latex having an average particle size of 0.3 μm (solid content: 10%
(W / V), manufactured by Sekisui Chemical Co., Ltd.) 1 mL and 0.036M
PBS and the mixture were stirred at 30 ° C. for 60 minutes. Next, 0.05MPB containing 1% by weight of BSA in this solution.
After adding S, the mixture was stirred at 30 ° C. for 60 minutes, and then washed by centrifugation at 4 ° C. for 20 minutes at 18,000 rpm. The washing operation was performed three times. B in the resulting precipitate
100 mL of 0.05M PBS containing 1% by weight of SA was added thereto, and the latex was suspended. The suspension was subjected to dispersion treatment using an ultrasonic crusher to give an anti-HBs antibody-sensitized latex having a solid content of 0.1% (W / V). A liquid was prepared. This was used as the second reagent. 3) Reagent for measuring HBs antigen The reagent for measuring HBs antigen is usually measured, that is, HBs antigen.
In the measurement of the antigen, the first and second reagents for measurement were usually used. In the neutralization test, that is, in the test for confirming the HBs antigen, the first and second reagents for neutralization were used. 4) Standard HBs antigen solution, 0, 50, 100, 300, HBs antigen as a sample standard for neutralization test
Human serum at a concentration of 500 IU / mL was used. As samples, HBs antigen-positive sera: A to E, and
False positive sera FJ were used. Here, false positive serum is
Serum collected from a healthy person, which was determined to be positive in a normal test. 5) Normal measurement method (1) Preparation of calibration curve 20 μL of the sample and 150 μL of the first reagent for normal measurement were mixed and stored at 37 ° C. as appropriate, and 150 μL of the second reagent was added and stirred. After this, the wavelength 750m after 1 minute and 5 minutes
m, and the difference is determined by the change in absorbance (ΔAb).
s). A Hitachi 7150 type automatic analyzer was used for the measurement. A standard curve was prepared in advance by measuring the standard product, and in the following measurement, the amount of HBs antigen in the sample was calculated by substituting the change in absorbance of the sample into the above-mentioned calibration curve. (2) Measurement of Sample The amount of HBs antigen was measured using the HBs antigen positive serum and the false positive serum as samples according to the above-mentioned method 5) to (1). The results are shown in Table 1. 6) Neutralization test method A neutralization test was carried out on a sample which was determined to be positive in the normal measurement of the above 5)-(2). The measurement was performed in the same manner as in 5)-(1) above, except that the HBs antigen-positive serum and the false-positive serum were used as samples, and the first reagent for normal measurement was replaced with the first reagent for neutralization. The results are shown in Table 1. The neutralization ratio in the table was calculated according to the following equation. ## EQU1 ## 7) Judgment of results Judgment of the results was made according to the following criteria. (1) Normal measurement / measured value is less than 8 IU / mL ... negative / measured value is 8 IU / mL or more ... positive (2) Neutralization test / neutralization rate is less than 50% ... negative / neutralization (Example 2) Application to HBs antigen measurement reagent-2 Preparation of the first reagent for normal measurement and the first reagent for neutralization in Example 1 is as follows. Except having performed, it carried out similarly to Example 1. The results are shown in Table 1. 1) Preparation of first reagent (buffer solution) (1) Preparation of first reagent for normal measurement PEG was added to 0.05 M PBS containing 1% by weight of BSA.
Was dissolved to a concentration of 1.0% by weight. To 1 L of the above solution, 8.0 μL of 0.036 MPBS was further added to prepare a first reagent for normal measurement. (2) Preparation of first reagent for neutralization PEG was added to 0.05M PBS containing 1% by weight of BSA.
Was dissolved to a concentration of 1.0% by weight. To 1 L of the above solution, an anti-HBs rabbit antibody (purified product, buffer: 0.036 MPBS) (protein concentration: 1.0 mg / m2)
L, specific antibody titer: 5300 IU / mL (measured with Mediace HBs antibody (manufactured by Sekisui Chemical Co., Ltd.))
L was added to prepare a first neutralizing reagent. [Table 1] (Comparative Example 1) A first reagent for normal measurement was prepared as follows, and the first reagent for neutralization in Example 1 was replaced with the first reagent for confirmation test described in 1) to (2) below. The procedure was performed in the same manner as in Example 1 except that the test was performed using one reagent. The results are shown in Table 2. 1) Preparation of First Reagent (Buffer Solution) (1) Preparation of First Reagent for Normal Measurement PEG was added to 0.05 M PBS containing 1% by weight of BSA.
Was dissolved to a concentration of 1.0% by weight to prepare a first reagent for normal measurement. (2) Preparation of First Reagent for Confirmation Test PEG was dissolved in a Na-HCl borate buffer (pH 9.15) containing 1% by weight of BSA to a concentration of 1.0% by weight, and the confirmation test was performed. A first reagent was prepared. Comparative Example 2 A first reagent for normal measurement was prepared as follows, and the first reagent for neutralization of Example 1 was replaced with the first reagent for neutralization test of 1)-(2) below. The procedure was performed in the same manner as in Example 1 except that the test was performed using one reagent. The results are shown in Table 2. 1) Preparation of First Reagent (Buffer) (1) Preparation of First Reagent for Normal Measurement PEG was added to 0.05M PBS containing 1% by weight of BSA.
Was dissolved to a concentration of 1.0% by weight to prepare a first reagent for normal measurement. (2) Preparation of first reagent for confirmation test In a Na-HCl borate buffer (pH 9.15) containing 1% by weight of BSA, NaCl was adjusted to a concentration of 0.5M, and PEG was added to 1.0%. By dissolving to a concentration of 1% by weight, a first reagent for confirmation test was prepared. [Table 2] In Examples 1 and 2, all five HBs antigen-positive sera showed a value of 8 IU / mL or more in a normal test, and the neutralization rate exceeded 50%. On the other hand, the false positive serum exceeded 8 IU / mL in the normal test in all five samples, but was determined to be negative because the neutralization rate was less than 50%. In Comparative Examples 1 and 2, all five HBs antigen-positive sera showed 8 IU / mL or more in a normal test, and the neutralization rate exceeded 50%, so that the samples were determined to be positive. However, as for the false positive sera, all five samples exceeded 8 IU / mL in the normal test, and four of the five samples also had a neutralization rate of 50% or more, and thus were determined to be positive. Since the present invention has the above-mentioned structure, it is possible to check whether the antigen and / or the antibody has been consumed without the trouble of transferring the sample to another container, which is very simple. A neutralization test of the antigen-antibody reaction can be performed.

Claims (1)

Claims: 1. A method for determining an antigen-antibody reaction in which an insoluble carrier carrying an antigen or an antibody is used to measure an antibody or an antigen corresponding to the carried antigen or the antibody, the method comprising: The reagent configuration for the use of two or more reagents consisting of a suspension of an insoluble carrier and a buffer, these reagents are mixed at the time of measurement, the measurement is performed twice, For a sample in which a reaction has occurred at the time of the first sample measurement, the buffer used for measurement in the reagent composition is replaced with a buffer containing the same antibody or antigen as that supported on the insoluble carrier, and then the second sample is used. Take a measurement and respond to the first reaction
A method for determining an antigen-antibody reaction, comprising determining whether the reaction is a specific reaction or a non-specific reaction by measuring the degree of decrease in the reaction in the buffer solution.