JPH06289025A - Nonspecific reaction prevention composition for blocking and solid-phase carrier - Google Patents

Abstract

PURPOSE:To achieve an accurate immunological measurement by blending tannic acid with a blocking composition. CONSTITUTION:Tannic acid has an operation for preventing nonspecific reaction in immunoreaction, especially that for preventing nonspecific reaction due to hemoglobin, and is blended with a blocking composition. For example, bovine serum albumin, zelatine, etc., are used as a blocking agent. A composition where the blocking agent and the tannic acid are used together can be used for every immunological measurement. For example, the competition method, the sandwich method, the liquid-phase method, and the solid-phase method are combined and used for the radiation enzyme measuring method and the enzyme immunoassay method, etc. Humor constituents such as serum, plasma, and fluid in the thoracic cavity are preferable as a specimen which can be used for immunoassay and enzyme, hormone, fetal protein, etc., are used as substances to be inspected.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a non-specific reaction-preventing composition for blocking, which prevents a non-specific reaction that adversely affects the measurement value in an immunological assay method and enables more accurate measurement. The present invention relates to a solid phase carrier on which is immobilized.

[0002]

2. Description of the Related Art Conventionally, as a method for measuring various substances in a biological fluid, there is an immunological measuring method utilizing an antigen-antibody reaction. Such immunological assay methods include enzyme-linked immunosorbent assay (EI
A), radioimmunoassay (RIA), etc., and these are extremely widely used because of their high sensitivity.

However, in these immunological assay methods, the reaction between an antigen and an antibody is extremely specific, and therefore, even a substance present in an extremely small amount in a sample can be assayed.
When the sample to be used is a biological sample such as serum, plasma, urine, etc., undesired non-specific reactions may also occur due to non-measured components such as various ions, complement, hemoglobin, etc. in the sample. There are many. Such non-specific reactions other than the intended antigen-antibody reaction have a great influence on the measured value when the amount of the component to be measured is small.

Therefore, various blocking agents have been developed in order to prevent these non-specific reactions. As typical ones, bovine serum albumin, gelatin, non-specific serum, casein, casein degradation product and the like are known.

[0005]

However, there are cases where sufficient blocking cannot be achieved even with these conventional blocking agents. For example, when blood that is not fresh blood is used as a sample, or when some occult blood exists in urine. The reason why a sufficient blocking effect is not obtained in such a case is not clear, but it is considered to be a non-specific reaction mainly due to hemolytic hemoglobin. Therefore, an object of the present invention is to provide a non-specific reaction-preventing composition for blocking in an immunological assay which does not depend on the type of biological sample and does not affect the reaction system.

[0006]

Therefore, as a result of various studies on the prevention of non-specific reaction in blood in which hemolysis has progressed as a sample, the present inventors have found that if tannic acid is added to a blocking composition, such non-specific reaction will occur. The inventors have found that the reaction can be effectively prevented, and that the addition of the tannic acid does not have any adverse effect on the antigen-antibody reaction, thus enabling accurate immunological measurement, and completed the present invention.

That is, the present invention provides a non-specific reaction-preventing composition for blocking, which comprises tannic acid. The present invention also provides a solid phase carrier comprising the blocking non-specific reaction-preventing composition immobilized on the carrier.

The term "non-specific reaction" as used herein means a non-specific reaction that occurs after blocking with an ordinary blocking agent. In the case of solid phase blocking, it is a non-specific reaction that occurs after the antibody is solid-phased and blocked, and is caused by the binding of the non-specific substance to the molecular gap of the blocking agent or the blocking agent itself. The tannic acid used in the composition of the present invention has a function of preventing a non-specific reaction in an immune reaction, particularly a function of preventing a non-specific reaction due to hemolyzed hemoglobin. As the tannic acid, a commercially available one can be used.

The content of tannic acid in the composition of the present invention is generally 0.005 to 0.2% by weight, preferably 0.01 to 0.1% by weight, although it cannot be generally stated. This blending amount is 0.
If it is less than 005% by weight, a sufficient effect cannot be obtained, and if it exceeds 0.2% by weight, tannic acid is precipitated,
This may adversely affect the measurement system and is not preferable.

The composition of the present invention is usually used by dissolving tannic acid in a buffer. The buffer solution is not particularly limited, but a phosphate buffer solution, a Tris-hydrochloric acid buffer solution and the like are preferable.

The blocking agent contained in the composition of the present invention is blended with one or more selected from bovine serum albumin, gelatin, non-specific serum, casein, casein degradation product, heat-treated casein and the like. Is preferred. The combined use of these conventional blocking agents and tannic acid further improves the blocking action as described later. Here, it is preferable to add 0.5 to 5% by weight, particularly 1% by weight, of bovine serum albumin to the total amount of the composition of the present invention, and 0.05 to 1.0% by weight, particularly 0.2 to 0% of gelatin. It is preferable to add 0.5% by weight. The non-specific serum is, for example, animal serum such as bovine at a temperature of 50 ° C. or higher for 15 minutes or longer, more preferably 50 to 60 ° C. for 15 to 6 sec.
It can be prepared by treating for 0 minutes, and it is preferable to add 0.5 to 5% by weight, particularly 1% by weight. Casein is preferably added in an amount of 0.1 to 2% by weight. The casein degradation product can be prepared by treating casein with a digestive enzyme such as chymotrypsin, papain or the like, for example, according to the description in JP-A-2-36353, and 0.05 to 2.5% by weight is added. Is preferred.
The heat-treated casein is, for example, 40-250 in casein solution.
It is prepared by heating to 150 ° C., preferably 60 to 150 ° C. for 15 to 45 minutes, and preferably 0.001 to 1000 mg / ml, particularly 0.05 to 10 mg / ml. These can be used alone or as a mixture.

The thus-obtained composition of the present invention can be used for any immunological assay, for example, RI combining a competitive method, a side-itch method, a liquid phase method, a solid phase method and the like.
It can be used for A, EIA and the like.

The blocking method using the composition of the present invention in these measuring methods is carried out by a conventional method, for example, the method described in "Monoclonal antibody" (translated by Toshiaki Osawa, published by Tokyo Kagaku Dojin). Specifically, for example, in the case of the solid phase method, a method in which a test substance or an antibody is chemically or physically bound to a solid phase carrier, and then the composition of the present invention is allowed to stand overnight is used.

In the immunoassay using the composition of the present invention, the sample that can be used is not particularly limited, but body fluid components such as serum, plasma, pleural effusion and urine are preferable.
The test substances include enzymes, hormones, fetal proteins (C
EA) and the like.

Further, the insolubilized test substance and the insolubilized antibody used in the immunoassay are produced by chemically or physically binding the test substance or antibody to the insolubilized carrier. Here, as the insolubilizing carrier, cellulose powder, sephadex, sepharose, polystyrene, filter paper, carboxymethyl cellulose, ion exchange resin, dextran, plastic film, plastic tube, nylon, glass beads, silk, polyamine-methyl vinyl ether-maleic acid copolymer Examples thereof include a combination, an amino acid copolymer, and an ethylene-maleic acid copolymer.
The insolubilization can be carried out, for example, by the diazo method as a covalent bond method, the peptide method (acid amide derivative method, carboxychloride resin method,
Carbodiimide resin method, maleic anhydride derivative method, isothianaate derivative method, cyanogen bromide activated polysaccharide method, cellulose carbonate derivative method, method using condensation reagent), alkylation method, carrier condensation method using crosslinking reagent (crosslinking reagent) As glutaraldehyde, hexamethylene isothiate, etc.), a chemical reaction such as carrier binding method by Ugi reaction; or an ionic bonding method using a carrier such as an ion exchange resin; porous glass such as glass beads as a carrier The physical adsorption method or the like is used.

[0016]

The action and effects of the invention The tannic acid contained in the composition of the present invention strongly prevents the non-specific reaction due to hemolyzed hemoglobin, but this action is particularly excellent in blocking the solid phase, and other blocking agents. It shows a remarkable effect when used in combination with, so by blocking the molecular gap of the blocking agent when blocking the solid phase with a blocking solution, or by preventing the non-specific substance from binding to the blogging agent itself, It is presumed that it has an action of preventing a specific reaction. Therefore, the composition of the present invention enables accurate measurement even with a hemolyzed sample, which has been difficult to measure conventionally, and can be used for all antigen-antibody reactions.

[0017]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0018]

[Table 1] The reagents and the like used in this example are shown below. AFP Purified Antigen Co., Ltd. Japan Biotest Laboratories Hemolysis hemoglobin (interference check) International Reagents Fetal bovine serum Mitsubishi Kasei Horseradish Peroxidase (HRP) SIGMA blocking agents Snow Brand Milk Products Co., Ltd. Block Ace (trade name) 96-well EIA microplate Nunc anti-AFP monoclonal antibody (Introduction to immunology experiments, produced by Nao Matsuura et al., Anti-AFP Polyclonal Antibody Publishing Center)

Example 1 α-fetoprotein (AFP) by the sandwich method
Enzyme immunoassay method: (1) AFP standard antigen was added to serum of a healthy subject at 2 ng / ml to prepare a simulated sample. In addition, 3 mg for this simulated sample
The hemolyzed hemoglobin was added to the mixture to give a hemolysis sample. (2) An anti-AFP polyclonal antibody solution dissolved in 0.1 M phosphate buffer (pH 7.0) at a concentration of 4 μg / ml was added to a 96-well EIA microplate at 2 times per well.
00 μl was dispensed and solidified overnight at 4 ° C. (3) 0.15M phosphate buffered saline (pH 7.2) 3
100 ml of Block Ace was mixed with 00 ml to obtain a blocking solution. To this, 0.02% by weight of tannic acid (0.08 g) was added to prepare a composition of the present invention.
After removing the anti-AFP polyclonal antibody solution from the wells, the composition of the present invention was added to the microplate at 40 / well.
0 μl was dispensed and blocking was performed at room temperature for 3 hours. (4) After removing the composition of the present invention, 50 μl each of the simulated sample and the hemolyzed sample prepared in (1) are added to another hole, and further 10
% Bovine serum, 500 with 0.1M phosphate buffer (pH 6.5)
100 μl of HRP-labeled anti-AFP monoclonal antibody prepared at ng / ml was added, and the mixture was reacted at room temperature for 1 hour, and then added to 0.1.
It was washed with 15M phosphate buffered saline (pH 7.2). Color developing solution [0.02% H ₂ O ₂ 1 mg / ml o-phenylenediamine-containing 0.1 M citrate phosphate buffer (pH 5.
0)] 100 μl was added thereto, and a color reaction was carried out for 30 minutes. The color reaction was stopped by mixing 100 μl of 1N H ₂ SO ₄ and colorimetric measurement was carried out at a wavelength of 492 nm. The results are shown in Table 2.

Example 2 The same operation as in Example 1 was carried out except that the amount of tannic acid added to the blocking solution was changed to 0.1% by weight. The results are shown in Table 2.

Comparative Example 1 The same operation as in Example was carried out except that tannic acid was not added to the blocking solution. The results are shown in Table 2.

[0022]

[Table 2]

Example 3 AFP standard antigen concentration in the simulated sample was 50 ng / ml, 100
Instead of ng / ml, the same operation as in Example 2 was performed. Table 3 shows the measurement results of the simulated sample.

Comparative Example 2 AFP standard antigen concentration in the simulated sample was 50 ng / ml, 100
Instead of ng / ml, the same operation as in Comparative Example 1 was performed. Table 3 shows the measurement results of the simulated sample.

[0025]

[Table 3]

As can be seen from the results in Table 1, by using the composition of the present invention, non-specific reaction can be effectively prevented even when a hemolyzed sample is used, and accurate measurement can be performed.
As can be seen from the above, this composition does not affect the reaction system and can be used for any antigen-antibody reaction, particularly for immunological assay using the antigen-antibody reaction.

Claims (2)

[Claims] 1. A non-specific reaction preventing composition for blocking, which comprises tannic acid. 2. A solid phase carrier obtained by immobilizing the non-specific reaction preventing composition for blocking according to claim 1 on a carrier.