JP2000329764A - Immunoassay and reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method capable of inhibiting non-specific reaction due to the complement in immunoassay. SOLUTION: In immunoassay using the antigen-antibody reaction of a substance to be measured in a specimen and a substance capable of generating the antigen-antibody reaction therewith, antigen-antibody reaction is performed in the presence of polyhydric phenol. By this constitution, non-specific reaction due to the complement coexisting in the specimen can be inhibited by the action of polyhydric phenol to enable the enhancement of measuring accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immunoassay and an assay reagent. More specifically, the present invention relates to an immunological assay used in the field of clinical testing and the like, which relates to an immunological assay capable of suppressing a non-specific reaction due to a contaminant in a reaction system and a reagent used therefor.

[0002]

2. Description of the Related Art In the field of clinical testing, etc., as a method for measuring a substance to be measured in a sample, an immunological reaction using an antigen-antibody reaction between the substance to be measured and a substance capable of reacting with the substance and an antigen-antibody has been conventionally used. Measurement methods are widely used. Examples of such a measuring method include an immunodiffusion method (SRID method), an immunoturbidimetric method, a hemagglutination method, a method using a carrier such as latex, an RIA method, and an EIA method. In such an immunoassay, in addition to the desired antigen-antibody reaction, aggregation or adsorption due to non-specific reactions with complements, immune complexes, anti-human mouse antibodies, lipids, and other contaminants in the sample occurs. Easy,
For this reason, there has been a problem that measurement accuracy and reliability are lacking. Conventionally, as a method for suppressing such non-specific reactions, there is known a method of adding a polyanion such as dextran sulfate, heparin, polystyrene sulfonic acid, or chondroitin sulfate to a reaction system.

[0003]

However, in the above-mentioned method using a polyanion, the viscosity of the reaction solution increases because the concentration of the polyanion must be increased in order to suppress the non-specific reaction. It had drawbacks such as being too high and increasing the background value. The present invention has been made in order to solve such disadvantages of the prior art, and as a result of diligent research conducted by the present inventors, it has been found that the problems of the conventional immunological assay method can be improved to improve the non-specific reaction. We have found a method that can be removed and completed it. That is, an object of the present invention is to provide an immunological measurement method which is excellent in measurement accuracy and can be usefully used in the field of clinical tests and the like.

[0004]

The immunological assay of the present invention which has been made to solve the above-mentioned problem is characterized by adding a polyhydric phenol, particularly a calixarene, to a reaction system. Further, the reagent for immunological assay of the present invention,
A reagent used for an immunological assay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, wherein the reagent contains a polyhydric phenol. It is characterized by the following. That is,
By adding polyhydric phenol (preferably calixarene) to the reaction system, non-specific reaction of contaminants such as complement is suppressed, and measurement accuracy is improved.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an immunoassay is particularly preferably an immunoassay using an antigen-antibody reaction between a substance to be measured and a substance capable of reacting with the substance and an antigen and antibody. Without limitation, for example, immunodiffusion (S
RID method), immunoturbidimetry, hemagglutination method, method using a carrier such as latex, RIA method, and EIA method.

The subject to be measured in the method of the present invention is not particularly limited as long as it is a substance to be measured by an immunological assay, and various substances (eg, antigens, haptens, antibodies, hormones, drugs, etc.) can be mentioned. . More specifically, for example, CRP, fibrin and fibrinogen degradation products, IgG, IgA, IgM, IgE, IgD, anti-strepridin O, rheumatoid factor, transferrin, haptoglobin, α1-antitrypsin, α1-acid glycoprotein, α2 -Macroglobulin, hemodoxin, antithrombin-III, α-fetoprotein, CEA (carcinoembryonic antigen), ferritin, HBs-Ag (hepatitis B envelope antigen), Anti-
HBs (anti-hepatitis B envelope), HBe-Ag (hepatitis B e
Antigen), Anti-HBe (anti-hepatitis B e), Anti
-HBc (anti-hepatitis B core) and the like. Samples (specimens) containing these substances include, for example, serum, plasma, urine, cerebrospinal fluid, and lymph.

The substance (hereinafter referred to as an immunoreactive substance) capable of antigen-antibody reaction with the above-mentioned substance to be measured is an antibody when the substance to be measured is an antigen, a hapten or the like, and an antibody when the substance to be measured is an antibody. An antigen is used. Antigens and antibodies as immunoreactive substances can be prepared by conventional methods. The antibody may be either a polyclonal antibody or a monoclonal antibody. Further, the F (ab) ₂ fraction,
The F (ab ') ₂ fraction may be used.

The polyhydric phenol used in the present invention includes, for example, catechin, quercetin, isoflavone, tannin, catechol, resorcinol, eugenol, calixarene, and the like. These polyhydric phenols may be used in combination of two or more. Is also good. In the above polyhydric phenols, calixarenees are preferably used, and the calixarenees are cyclic oligomers having phenol as a basic skeleton and 4 to 8 molecules of phenol polymerized cyclically with methylene groups. The calixarenes are not particularly limited, and various types can be used. For example, calix (4) arene [Calix (4) arene], calix (6) arene, calix (8) arene, calix sulfate (4) Allene, calix sulfate (6) Allene, calix sulfate (8) Allene, calix acetate (4) Allene, calix acetate (6)
Allene, calix acetate (8) allene, carboxy calix (4) allene, carboxy calix (6) allene, carboxy calix (8) allene, calix (4) allenamine, calix (6) allenamine, calix (8) )
Allenamine and the like are exemplified, and calix sulfate (6) arene and calix sulfate (8) arene are preferable. Two or more of these calixarenees may be used in combination. The concentration of the polyhydric phenol in the reaction system is adjusted to about 0.05 to 50 mM, preferably about 0.1 to 20 mM before use. Further, conventional additives such as polyethylene glycol, dextran, gelatin, sodium chloride, and EDTA may be added to the reaction system as needed.

An example of the method of the present invention will be described with reference to an example in which CRP in a sample is measured by an immunoturbidimetric method. First, a polyhydric phenol is added to an appropriate buffer (for example, HEPES buffer, about pH 7.5). (Preferably calixarene) so that the final concentration is 0.05 to 50 mM,
If necessary, polyethylene glycol, EDTA,
A first reagent to which an additive such as sodium chloride is appropriately added is prepared. On the other hand, an anti-CRP antibody (for example, anti-human CRP goat serum or the like) is added to an appropriate buffer (for example, HEPES buffer, about pH 7.5). And a second reagent to which additives such as polyethylene glycol and sodium chloride are appropriately added as needed is prepared. After appropriately diluting a sample containing CRP (for example, serum or the like) with the first reagent, the sample is cooled to about 20 to 40 ° C. (generally, about 37 ° C.).
Heat for about 10 minutes (usually about 5 minutes). Then
Add an appropriate amount of the second reagent, and add about 20 to 40 ° C (usually,
(About 37 ° C) for about 2 to 10 minutes (usually about 5 minutes)
After heating, the absorbance at a wavelength of 340 nm is measured, and the CRP concentration can be determined based on a previously prepared calibration curve. The measurement of the absorbance may be performed by any of the end point method and the rate assay method. Note that the method of the present invention is not limited to the above method, and can be carried out by appropriately changing the method according to the immunological assay method to be used. do it.

[0010] The reagent of the present invention is a reagent used in the above method, which reagent contains a polyhydric phenol. The above reagents can be appropriately set according to the type of the immunological assay, and may be a kit comprising a plurality of constituent reagents. In this case, at least one of the constituent reagents is a polyhydric phenol. May be contained.

[0011]

According to the method of the present invention, nonspecific reactions due to contaminants such as complements in a sample are suppressed by polyhydric phenol, and polyhydric phenol can increase the viscosity of the reaction solution. The reactivity does not become too high. Therefore, according to the method of the present invention, there is an effect that the measurement accuracy of the immunological assay can be significantly improved. Further, the reagent of the present invention contains a polyhydric phenol. According to the reagent of the present invention, a reagent capable of exhibiting the above effects can be obtained.

[0012]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 In order to measure CRP in human serum, 10 human sera were used as samples. Then, 250 μL of the following first reagent was added to 15 μL of human serum, and the mixture was heated at 37 ° C. for 5 minutes, and then 50 μL of the following second reagent was added.
After heating for minutes, the absorbance at a wavelength of 340 nm was measured,
A standard curve with a known CRP concentration was converted into a CRP value from a calibration curve prepared in advance by the same operation (referred to as measurement A). On the other hand, calix sulfate (6) allene 5 was used as the first reagent.
A solution to which mM is added is prepared as a third reagent. This third
Except that the reagent was used instead of the first reagent, the operation was performed in the same manner as in Measurement A, and converted into a CRP value (referred to as Measurement B). Furthermore, the same sample as the human serum sample used in Measurements A and B was heated in advance at 56 ° C. for 30 minutes to immobilize it, and the complement component was inactivated. (Referred to as measurement C). Table 1 shows the results obtained by the measurement methods A to C.

First reagent 50 mM HEPES buffer pH 7.5 4% Polyethylene glycol 6000 1% NaCl 1 mM EDTA Second reagent 50 mM HEPES buffer pH 7.5 1% NaCl 15% Anti-human CRP rabbit serum (manufactured by Nagase & Co., Ltd.)

[0014]

[Table 1]

As shown in Table 1, the measured value of the measurement B is lower than that of the measurement A, and the measured values of the measurements B and C are almost the same. Therefore, in the present invention, a calixarene is added to the reaction system. It was found that the method (measurement B) can suppress non-specific reactions.

Claims (3)

[Claims] In an immunoassay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, the reaction is performed in the presence of a polyhydric phenol. Characteristic immunoassay. 2. The immunoassay according to claim 1, wherein the polyhydric phenol is a calixarene. 3. A reagent for use in an immunological assay using an antigen-antibody reaction between a substance to be measured in a sample and a substance capable of reacting with the substance to be measured and an antigen-antibody reaction, wherein the reagent is a polyvalent reagent. A reagent for an immunoassay, comprising phenol.