JP2015184125A - Immunoassay suppressing non-specific reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method suppressing a non-specific reaction generated in an immunoassay.SOLUTION: The immunoassay for measuring an antigen or antibody in a specimen, when the antigen and antibody are reacted, causes a basic amino acid such as lysine, arginine, or histidine, basic amino acid salt, peptide or protein (e.g., polymer, histone, or protamine of the basic amino acid) with a ratio of the basic amino acid of 10% or more to coexist therein so as to suppress the non-specific reaction.

Description

  The present invention is characterized in that, in an immunological measurement method for measuring an antibody or antigen in a sample, a basic amino acid, a basic amino acid salt, or a peptide or protein having a basic amino acid ratio of 10% or more coexists. It is about the method.

  The immunological measurement method is used for analysis, measurement, quantification, detection, and the like of a measurement object, and is widely used particularly in a medical field as a clinical test drug. This immunological measurement method uses an antigen-antibody reaction between an antigen and an antibody against the antigen, but various techniques have been developed to facilitate the detection and detection thereof. Specifically, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay, agglutination immunoassay and the like have been developed as immunological assays and are currently widely used.

The antibody or antigen involved in the antigen-antibody reaction is often bound to an insoluble carrier or labeled by a detectable method. For example, as an insoluble carrier, synthesis of bio-derived polymers such as agarose, nitrocellulose, dextran or polymers derived from natural products, acrylic resins such as polystyrene, polyethylene, polyvinyl alcohol, polypropylene, polyacrylamide, Teflon (registered trademark), polyacetal, etc. Examples thereof include fine particles, beads, microplates, microtiter wells, microtubes, strips, membranes, gels, and the like made of inorganic materials such as polymers, glass, silica gel, alumina, ceramics, carbon, and magnesium sulfate. The antigen or antibody immobilized on the insoluble carrier is specifically bound to the antibody or antigen in the analysis sample to detect the antibody or antigen that is the specifically bound analyte. Examples of detectable methods include radioactive substances such as ¹²⁵ I and ³ H, horseradish peroxidase, β-D-galactosidase, enzymes such as alkaline phosphatase, fluorescent substances such as fluorescein, luminescence of gold colloid, selenium colloid, etc. Alternatively, an antigen or antibody labeled with a coloring substance or the like is used as a reagent.

  Such immunoassay is widely used in clinical practice as a clinical test agent, but detection of HBs antigen and HCV antibody for confirming the presence of infection such as hepatitis B virus and hepatitis C virus. In the diagnosis of cancer using a tumor marker or the like, when the determination is made based on the cut-off value, it is a fatal problem in the medical field that the measured value shows a deviation value.

  Such divergence values are due to non-specific reactions that occur between the antigen or antibody used in the immunoassay and the components in the measurement sample. Conventionally, the surface of the insoluble carrier or the free binding site of the labeled antibody Although it has been proposed to use a blocking agent for blocking, since it is not sufficient, it is strongly required to eliminate the divergence value caused by non-specific reaction.

  As described above, immunological measurement methods are widely used in the medical field, but sometimes misjudgment is caused by deviation values caused by non-specific reactions. In particular, immunoassays used to detect infectious disease antibodies such as HCV antibodies, HBs antibodies, and TP (Treponema pallidum) antibodies often use recombinant antigens as well as natural antigens. A non-specific binding reaction with an antibody other than the above antibody may occur. In the past, as a blocking agent for blocking the surface of the insoluble carrier and the free binding site of the labeled antibody in order to minimize non-specific interactions, proteins such as bovine serum albumin, casein, gelatin, Triton X A method using a surfactant such as −100 and Tween 20 has been used. However, nonspecific reactions still remain a problem in immunoassay technology. An object of the present invention is to provide a method for suppressing a nonspecific reaction occurring in an immunoassay.

  As a result of intensive studies, the inventor has added a basic amino acid, a basic amino acid salt, or a peptide or protein containing a large amount of basic amino acid in an immunoassay for detecting an antibody or antigen in a sample. The present inventors have found that a specific reaction can be suppressed and have reached the present invention.

That is, the present invention is as follows.
(1) In an immunological assay for measuring an antigen or antibody in a sample, a peptide or protein having a basic amino acid, basic amino acid salt, or basic amino acid ratio of 10% or more when the antigen and antibody react A method characterized by coexisting.
(2) The method according to (1), wherein the basic amino acid is lysine, arginine or histidine.
(3) The method according to (1), wherein the basic amino acid salt is lysine hydrochloride, arginine hydrochloride or histidine hydrochloride.
(4) The method according to (1), wherein the peptide or protein having a basic amino acid ratio of 10% or more is a basic amino acid polymer, histone or protamine.

  The present invention is described in further detail below.

  In the present invention, a basic amino acid, a basic amino acid salt, or a peptide or protein having a basic amino acid ratio of 10% or more (hereinafter also referred to as a non-specific reaction inhibitor) is generally commercially available, alone or in combination. Can be used. The amount of the nonspecific reaction inhibitor used in the present invention is preferably 0.1 to 10 w / v%, more preferably 0.5 to 5 w / v% in the immune reaction solution.

  Although there is no limitation in particular as a basic amino acid, Preferably it is a lysine, arginine, or histidine. The basic amino acid salt is not particularly limited, and hydrochloride, nitrate, sulfate and the like are used, and hydrochloride is preferable. A peptide or protein having a basic amino acid ratio of 10% or more means a peptide or protein having a high basic amino acid content, and examples thereof include basic amino acid polymers, histones, and protamines.

  In the present invention, a label can be used. As the label, for example, any enzyme, fluorescent substance, radioisotope, etc. are effective. Alternatively, these substances may be directly labeled using biotin-avidin or the like without being labeled with a substance used for detection. For example, a known enzyme immunoassay (EIA, ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), or luminescent immunoassay (LIA) can be used to detect a labeled substance bound to an antigen. Can be performed.

  In the present invention, an insoluble carrier can be used. As for the insoluble carrier, beads, tubes, plates, magnetic fine particles, etc. made of well-known substances such as glass, polystyrene, polypropylene, dextran, etc. can be used. It doesn't matter. The insoluble carrier and the antibody (or receptor or binding protein) are preferably bound by a method that does not lose the binding ability during B / F separation, such as physical binding or chemical binding via an intermediate.

  According to the present invention, a basic amino acid, a basic amino acid salt, and a peptide containing a large amount of basic amino acid or a protein containing a large amount of basic amino acid are allowed to coexist and immunology can be performed without using a so-called blocking agent. It becomes possible to suppress the nonspecific immune reaction that occurs in the measurement. The non-specific reaction inhibitor used in the present invention is generally commercially available and is a well-known material, and can be used alone or in combination.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by a present Example.

Example 1
Using an AIA-1800 automatic immunoassay device (manufactured by Tosoh Corporation) as an immunoassay device, TP antibodies were measured by a one-step competition method. Specifically, first, ferrite-containing ethylene vinyl acetate copolymer beads immobilized with three kinds of TP antigens (15 kDa, 17 kDa, 47 kDa) and a primary reaction solution (1% BSA 200 mM HEPES buffer (pH 7)) are dispensed into a reaction cup. And lyophilized to prepare Reagent A. Further, Reagent F was prepared from Reagent B to which 0.1, 1.0, 5.0, 10 w / v% lysine hydrochloride or 5.0 w / v% arginine hydrochloride was added to the primary reaction solution under the same conditions.

  In the measurement, first, a test sample (serum) and a lyophilized solution were simultaneously added to a reaction cup of Reagents A to F, and reacted at 37 ° C. with stirring for 10 minutes. Thereafter, the unreacted product was removed by B / F separation, and a secondary reaction solution (50 mM phosphoric acid containing 1% BSA) containing an anti-human immunoglobulin mouse monoclonal antibody (manufactured by Tosoh AIAI) labeled with alkaline phosphatase. Buffer solution (pH 7)) was dispensed and reacted at 37 ° C. for 10 minutes with stirring. Thereafter, unreacted substances were removed by B / F separation, and a substrate solution containing 4-methylumbelliferyl phosphate was added. The production (nM / sec) of 4-methylumbelliferone per unit time produced by alkaline phosphatase-labeled anti-human immunoglobulin mouse monoclonal antibody bound to TP antigen via TP antibody was measured. This production rate is proportional to the amount of alkaline phosphatase, and the concentration of TP antibody in the test sample can be known from the intensity of fluorescence.

  The TP antibody concentration in the sample was determined after preparing a calibration curve in advance using a TP antibody standard. The determination of the measurement result of the TP antibody was determined as positive (+) at 1.0 COI (cutoff index) or more and negative as less than 1.0 COI.

  Using the prepared reagents A to F, three TP antibody negative samples and three TP antibody positive samples were measured. The TP antibody negative specimen is collected from a blood donor who has no history of syphilis.

  Table 1 shows the results of measuring TP antibody negative and positive specimens. As can be seen from Table 1, the false positive sample that showed a value of 1.0 COI or higher when measured with Reagent A is lower with Reagent B, and Reagent C to Reagent E show values of less than 1.0 COI. And converged around 0 COI when the lysine concentration was 5 w / v% or more (reagents D and E). By adding lysine hydrochloride to the primary reaction solution, the non-specific reaction due to the components in the negative sample was reduced, and there were no false positive samples. In addition, even with reagent F, there was a tendency for non-specific reactions to be low. Although it did not decrease as much as reagent C to which the same concentration (1 w / v%) was added, the addition of arginine hydrochloride was also effective.

  On the other hand, in the TP antibody positive sample, the reagent A to the reagent F show similar values, and the reactivity of the positive sample is not lowered even when a basic amino acid is added to the primary reaction solution, and the TP antibody is measured. Was able to.

  Judging from the above results from Reagent A to Reagent F, it is clear that basic amino acids avoid non-specific reactions of TP antibody negative samples. It was found that the effect of lysine hydrochloride was particularly high.

実施例２
実施例１で作製された試薬Ａと試薬Ｄを用いて１３例のＴＰ抗体陰性検体と８例のＴＰ抗体陽性検体を測定した。ＴＰ抗体陰性検体は、梅毒の病歴がない供血者から採取したものである。

Example 2
Using Reagent A and Reagent D prepared in Example 1, 13 TP antibody negative samples and 8 TP antibody positive samples were measured. The TP antibody negative specimen is collected from a blood donor who has no history of syphilis.

  Table 2 shows the results of measuring 13 TP antibody negative specimens. As can be seen from Table 2, the false positive sample that showed a value of 1.0 COI or higher when measured with Reagent A began to show a value of less than 1.0 COI with Reagent D, and was totally dispersed. It can be seen that the value converges around 0 COI. By adding lysine hydrochloride to the primary reaction solution, the non-specific reaction due to the components in the negative sample becomes very small and there are no false positive samples.

  Table 3 shows the results of measuring 8 TP antibody positive specimens. Both Reagent A and Reagent D showed similar values, and even when lysine hydrochloride was added to the primary reaction solution, the reactivity of the positive sample was not lowered and the TP antibody could be measured.

  Judging from the results of the above reagent A and reagent D, it is clear that basic amino acids avoid non-specific reactions of TP antibody negative specimens.

Claims (4)

In an immunological assay for measuring an antigen or antibody in a sample, when an antigen and an antibody react, a basic amino acid, a basic amino acid salt or a peptide or protein having a basic amino acid ratio of 10% or more is allowed to coexist. A method characterized by that. The method according to claim 1, wherein the basic amino acid is lysine, arginine or histidine. The method according to claim 1, wherein the basic amino acid salt is lysine hydrochloride, arginine hydrochloride or histidine hydrochloride. The method according to claim 1, wherein the peptide or protein having a basic amino acid ratio of 10% or more is a polymer of a basic amino acid, histone or protamine.