JP2015184137A - immunoassay method and immunoassay reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of promoting a reaction between an antigen and an antibody without greatly increasing the background.SOLUTION: Under the coexistence of 1-50 mM amino acid (preferably, an asparaginic acid or glutamic acid as an acidic amino acid), an antigen or antibody in a sample is made to react with an antibody or antigen immobilized in an insoluble carrier. The immunoassay reagent includes an antibody or antigen immobilized in the insoluble carrier and an amino acid, and is freeze-dried.

Description

  The present invention relates to an immunoassay method and an immunoassay reagent. More specifically, in the present invention, when reacting an antigen or antibody in a sample with an immobilized antibody or antigen, an amino acid is allowed to coexist so that the background between the antigen and the antibody is not significantly increased. The present invention relates to a method for improving reactivity.

  An insoluble carrier on which a biological substance is immobilized has been developed and used as a diagnostic agent material for measuring various physiologically active substances. In particular, an insoluble carrier on which an antibody is immobilized is extremely useful as a diagnostic agent material for immunoassay and is widely used.

  As the above insoluble carrier, an immunoassay using an insoluble carrier on which an antibody that recognizes a measurement target is immobilized has been developed and is generally used. In the immunoassay, for quantification of the measurement target, the difference between the value (S) for detecting the measurement target and the value (N) when there is no measurement target is sufficiently large, that is, the S / N needs to be large. .

  There is a method of coexisting a buffer solution containing protein and sugar in order to promote the reaction and improve the accuracy and reproducibility of the value in the immune reaction between the measurement target and the antibody against it. Therefore, the above components alone are not sufficient, and there is room for further improvement.

  Accordingly, an object of the present invention is to provide a method for promoting the reaction without greatly increasing the background by allowing an amino acid to coexist during the reaction between an antigen and an antibody.

In order to solve the above-mentioned problems, the present inventors have intensively studied and as a result, have reached the present invention. That is, the present invention is as follows.
(1) An immunoassay method comprising reacting an antigen or antibody in a sample with an antibody or antigen immobilized on an insoluble carrier in the presence of an amino acid.
(2) The method according to (1), wherein the amino acid is an acidic amino acid.
(3) The method according to (1) or (2), wherein the amino acid is aspartic acid or glutamic acid.
(4) The method according to any one of (1) to (3), wherein the concentration of the amino acid is 1 to 50 mM.
(5) An immunoassay reagent comprising an antibody or antigen immobilized on an insoluble carrier, and an amino acid.
(6) The immunoassay reagent according to (5), which is freeze-dried.

  Hereinafter, the present invention will be described in more detail.

In the present invention, the antigen or antibody in the sample to be measured is not particularly limited, and examples of the antigen include thyroglobulin, TSH, CEA, CA125, CA19-9 and the like. Insoluble carrier is not particularly limited as antibodies or antigens are immobilized, monoclonal antibodies and polyclonal antibodies can be cited to the antigen mentioned above, also like FT ₃ may be mentioned as an antigen. These do not necessarily have to be derived from humans, and may be derived from, for example, rats, pigs, dogs, and rabbits. Furthermore, in addition to extracted and purified products derived from living tissues, chemical synthetic products, products produced by gene recombination techniques, and the like may also be used.

  In the present invention, the antibody or antigen is immobilized on an insoluble carrier and used. As the insoluble carrier, for example, a plate made of glass, resin, metal or the like, particles, fine particles, magnetic fine particles and the like are used. Among them, the effect of the present invention is remarkable for fine particles, magnetic fine particles, and the like. The method for immobilizing an antibody or antigen on an insoluble carrier is not particularly limited, and examples thereof include adsorption by a hydrophobic action and covalent bonding via a functional group on the surface of a fine particle.

  In the present invention, when reacting an antibody or antigen immobilized on an insoluble carrier with an antigen or antibody in a sample, an amino acid is allowed to coexist. The amino acid at this time is not particularly limited and may be any of acidic, neutral and basic amino acids, but is preferably an acidic amino acid, more preferably aspartic acid or glutamic acid. The concentration at which the amino acid is allowed to coexist is not particularly limited, but is preferably 1 to 50 mM, and more preferably 10 to 30 mM. These amino acids may be used alone or in combination of two or more. When using 2 or more types, it is preferable that the density | concentration of each amino acid exists in the above-mentioned range.

  In the present invention, protein or sugar may be allowed to coexist when the antibody or antigen immobilized on the insoluble carrier is reacted with the antigen or antibody in the sample. The protein is not particularly limited. For example, normal serum protein of mammals, albumin, collagen, gelisate, skim milk, lactic acid fermented product, gelatin and degradation products thereof can be used, and the concentration is not particularly limited. 1 to 50% is preferable. The sugar is not particularly limited, and for example, D-mannitol, sucrose, myo-inositol, trehalose, β-cyclodextrin, glucose, lactose, fructose, cellulose, raffinose, maltose, galactose, xylose, and the like can be used. The concentration is not particularly limited, but is preferably 1 to 10%, for example.

  In addition, protective agents (for example, bovine serum albumin), antioxidants (for example, ascorbic acid, vitamin E), binders (for example, carboxymethylcellulose), wetting agents (for example, cellulose, polyethylene glycol, etc.) ), Suspending agents (such as polyvinylpyrrolidone), emulsifiers (such as alkyl sulfonic acid), solubilizing agents (such as glycerin), buffers (such as phosphate, tris-hydroxylamine hydrochloride), isotonic An agent (for example, D-sorbitol, sodium chloride, etc.), magnesium chloride, zinc chloride, a surfactant (for example, Triton X-100, Tween 20, etc.) and the like may coexist.

  When performing the immunoassay of the present invention, an immunoassay reagent containing an antibody or antigen immobilized on an insoluble carrier and an amino acid may be used. This reagent may be lyophilized.

  According to the present invention, by coexisting amino acids, it is possible to promote the reactivity between an antigen and an antibody, that is, to improve the value detected by an immunoassay. At this time, since the background does not increase or is small, the S / N ratio can be increased.

It is a figure which shows the relationship between the presence or absence of the amino acid addition in Example 1, and a measured value. It is a figure which shows the relationship between the presence or absence of the amino acid addition in Example 1, and a measured value.

  Specific embodiments of the present invention will be described by way of examples. However, the present invention is not limited to these examples. In the examples, the immunoassay reagent provided by the present invention was measured as a sample for enzyme immunoassay (EIA) in the form of a lyophilized product, but the present invention shows a radioimmunoassay (RIA). It can be applied to all methods such as fluorescence immunoassay (FIA) or luminescence immunoassay (LIA).

Example 1 CEA immunoassay (1) Preparation of solid phase 1% (w / v) of magnetic microparticles (Dynabeads: manufactured by Dynal) as a solid phase in 0.01 mol / L MES buffer (pH 6.0) ) Anti-CEA antibody was added to the suspension having a slurry concentration to a concentration of 0.2 mg / mL and incubated at 37 ° C. for 3 hours to adsorb the antibody. After washing the fine particles, blocking was performed at 37 ° C. for 17 hours with a blocking solution containing 10% PEG as a raw material to obtain anti-CEA antibody-immobilized magnetic fine particles (A). Further, (A) was diluted with a 0.05 mol / L Tris buffer solution (pH 8.0) containing 5% bovine serum, and the amino acid described in Table 1 was added to prepare a solid phase suspension. Was lyophilized to obtain an immobilized antibody reagent (B).

(2) Detection labeled antibody By lyophilizing a conjugate of anti-CEA antibody and alkaline phosphatase diluted with 0.05 mol / L Tris buffer (pH 8.0) containing 5% collagen peptide, A labeled antibody reagent (C) was prepared.

(3) Preparation of standard solution A known concentration of CEA is adjusted to a predetermined concentration (0 ng / mL, 550 ng / mL) with 0.03 mol / L Tris buffer solution (pH 8.0) containing 5% collagen peptide. It diluted and it was set as the standard solution (D) and (D ') for CEA measurement.

(4) Measurement of standard solution Measurement was performed by chemiluminescent enzyme immunoreaction. In the measurement, the immobilized antibody reagent (B) was dissolved in pure water containing a surfactant and a standard solution (D or D ′), and an immune reaction was carried out at 37 ° C. for 5 minutes. Thereafter, B / F separation was performed. Next, a labeled antibody reagent (C) dissolved in pure water containing a surfactant was added, an immune reaction was performed at 37 ° C. for 3 minutes, and B / F separation was performed again. Thereafter, a chemiluminescent substrate for alkaline phosphatase (5-t-butyl-4,4-dimethyl-1- (3′-phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0] heptanedi Sodium salt), fluorescein, and tri-n-butylhexadecylphosphonium bromide were added, and the emission intensity (Count / sec.) Was measured. The results are shown in Table 1, Table 2, FIG. 1 and FIG.

これらの結果から明らかなように、アミノ酸を共存させることにより、抗原と抗体の反応性が促進され、検出値が大きく向上したが、バックグラウンドは上昇することはなく、結果としてＳ／Ｎ比が大きく向上した。この効果は酸性アミノ酸を用いた方が大きく、特にアスパラギン酸とグルタミン酸を併用した方が大きかった。

As is clear from these results, the coexistence of amino acids promoted the reactivity between the antigen and the antibody, and the detection value was greatly improved, but the background did not increase, and as a result, the S / N ratio was increased. Greatly improved. This effect was greater when acidic amino acids were used, especially when aspartic acid and glutamic acid were used in combination.

Claims (6)

An immunoassay method comprising reacting an antigen or antibody in a sample with an antibody or antigen immobilized on an insoluble carrier in the presence of an amino acid. The method of claim 1, wherein the amino acid is an acidic amino acid. The method according to claim 1 or 2, wherein the amino acid is aspartic acid or glutamic acid. The method according to any one of claims 1 to 3, wherein the concentration of the amino acid is 1 to 50 mM. An immunoassay reagent comprising an antibody or antigen immobilized on an insoluble carrier, and an amino acid. The immunoassay reagent according to claim 5, which is freeze-dried.

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