JP2001289850A - Method and reagent for immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which a substance to be inspected is measured specifically even when a cross-reacting substance coexists in a sample in the method in which the substance to be inspected is measured immunologically and to provide a measuring reagent. SOLUTION: In the method, the substance to be inspected is measured immunologically. In the immunoassay method, a means by which the reactivity of the coexisting cross-reacting substance is reduced is introduced, in detail, a substance which reduces the reactivity of the coexisting cross-reacting substance is made to coexist.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention is used as a test reagent for measuring a substance in a biological sample in the clinical test field.

[0002]

2. Description of the Related Art Measurement of a test substance in a sample using an immunological reaction is routinely performed. The measurement is performed according to various measurement principles depending on the concentration of the test substance. Among them, immunoturbidimetry, latex turbidimetry, enzyme immunoassay and the like are typical methods. Antibodies used for immunological reactions are prepared using a test substance as an antigen, and generally show a cross-reactivity with a similar substance of the antigen. Therefore, when a test substance and its analogous substance, ie, a cross-reactive substance, coexist in a sample, accurate measurement results cannot be obtained if the antibody used for immunological measurement has high cross-reactivity. Therefore, generally, an antibody having low cross-reactivity is selected to construct an appropriate measurement system.

When measuring the concentrations of thrombin and antithrombin-III complex (TAT), thrombin and antithrombin-III (AT-
III) There is known a method of measuring by a sandwich enzyme immunoassay using antibodies specific to each. In order to measure this TAT by latex immunoturbidimetry, it is necessary to prepare an antibody that is specific to TAT and does not cross-react with thrombin, prothrombin and AT-III. However, whereas the TAT concentration in plasma is usually about 1 to 50 ng / mL, AT-II
The concentration of I was about 250 μg / mL and 5,000-250,
000 times higher. Therefore, it is TAT-specific and AT-I
A monoclonal antibody having a cross-reactivity with II or less than 1 / 250,000 must be prepared. However, preparation of such a monoclonal antibody is difficult, and a reagent for measuring TAT by latex immunoturbidimetry is currently in practical use. Not.

[0004] On the other hand, when an antibody titer to a pathogen or the like generated in a biological sample is measured by an immunoagglutination reaction such as hemagglutination, it is necessary to check whether the antibody is IgG type or IgM type. Is treated with a reducing agent such as dithiothreitol to reduce the antibody activity of the IgM-type antibody, and the presence of the IgM-type is confirmed from the degree of reduction in the resulting aggregation. However, in the method of measuring an antigen, it is inconceivable to specifically measure a test substance by chemically treating contaminating cross-reactive substances.

[0005]

DISCLOSURE OF THE INVENTION The present invention relates to a method for immunologically measuring a test substance, and more particularly, to a method for specifically measuring a test substance even when a cross-reactive substance coexists in a sample. It is to provide a measurement reagent.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have introduced a means for reducing the reactivity of a cross-reactant contaminating in a sample. The present inventors have found that the presence of a substance that reduces reactivity in an immunoreactive system allows immunological measurement of a target test substance, which has been impossible so far, and completed the present invention.

[0007]

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immunologically measuring a test substance, which comprises introducing means for reducing the reactivity of contaminating cross-reactants. This is an immunoassay method characterized by the presence of a substance that reduces the amount of lipase.

In the present invention, as a substance capable of reducing cross-reactivity, a substance which cleaves a disulfide bond or a surfactant is effective. Specifically, dithiothreitol, mercaptoethylamine, glutathione, mercaptoethanol, monothiophosphoric acid, sodium borohydride, dithioerythritol, thioglycolic acid, dithionite, tris (2-carboxyethyl) phosphine, sulfite, α-thioglycerol, 2-aminoethylisothiouronium bromide hydrobromide and other substances that cleave disulfide bonds, as well as acids, alkalis, chaotropic compounds, water-soluble organic solvents, surfactants, etc. It is not limited to these. Further, these disulfide bond-cleaving substances may be further stabilized using a chelating agent such as EDTA. One or two of these substances
More than one kind can be used in appropriate combination. The concentration used can be easily determined by comparing the cross-reactivity of the test substance with the cross-reactive substance in the presence of these substances using the antibody used for the measurement of the test substance.

The means for reducing the reactivity of contaminating cross-reactive substances is preferably achieved by the addition of a specific substance as described above, but based on the knowledge of immunological reactivity,
For example, it is also possible by adjusting the ionic strength of the reaction solution, adjusting the pH, adjusting the reaction temperature, and the like. That is, if the cross-reactive substance in the sample is known, those skilled in the art can simply use known means that affect the immunological reactivity in order to reduce the immunological reactivity (antigen-antibody reactivity) of the substance. Such experimental repetition makes it possible to easily apply the present invention using the level of specificity of the substance as an index.

The substance which can be measured by the immunoassay method of the present invention includes substances which cannot be accurately measured due to cross-reacting substances contaminating in a sample, for example, a thrombin-thrombin inhibitor complex, a plasmin-plasmin inhibitor complex. , Thrombin and hirudin complex, elastase and α
1-antitrypsin complex, trypsin and trypsin inhibitor complex, activated factor X and AT-III complex, protein C and protein C inhibitor complex,
Tissue plasminogen activator and plasminogen activator inhibitor complex, activated factor VII and tissue factor pathway inhibitor (TFPI), tissue factor and coagulation factor VII complex, plasminogen and plasminogen activator complex, hemoglobin and haptoglobin complex, protein A complex such as A or protein G and an immunoglobulin complex can be exemplified.

The test substance is not limited to these, and can be used for any substance in which a cross-reacting substance coexists in a sample in a method for immunoassay of a test substance. That is, the immunoassay method of the present invention is useful for the measurement of these complexes, and also allows the coexistence of a substance capable of reducing the cross-reaction when the cross-reaction cannot accurately measure the substance originally intended for the measurement. Applicable to substances that can adopt the method.

Specifically, thrombin-
A preferred example is a thrombin inhibitor complex where the contaminating cross-reactants are thrombin inhibitor and / or
Or prothrombin. A typical example of a thrombin inhibitor is AT-III.

As will be described in detail in the following examples, a latex reagent in which a latex particle is adsorbed with a monoclonal antibody having a cross-reactivity with AT-III of about 1/1000 is prepared. The plasma sample is mixed and the aggregation process of the latex is measured optically. In the plasma sample, AT-III contained 5,000 to 5,000 of the test substance TAT.
Since there are 250,000 times more, this reagent
-III and about 5 times (250 ng / m 2) the upper limit of the abnormal value (50 ng / mL) even if no TAT molecule is present.
L) also gives a signal. However, if the reactivity is reduced to about 1/100 or less by adding a substance that reduces the immunological reactivity of the contaminating cross-reactive substance of the present invention, the measured value of 2.5 ng / mL or less can be obtained. Thus, a clinically useful test and measurement method can be provided.

In the examples, in the measurement of a thrombin-thrombin inhibitor complex, dithiothreitol (DTT) is used as an example of a substance that reduces the reactivity of AT-III.
However, the above substances can of course be used as appropriate.

Examples of immunoassays to which the present invention can be applied include enzyme immunoassay (EIA), nephelometry using a carrier, immunoturbidimetry without a carrier, immunochromatography, hemagglutination And other known immunoassays.

Further, the present invention provides a measurement reagent kit used in the above-mentioned immunoassay.

[0017]

【Example】

Example 1 A monoclonal antibody against TAT (clone No. TAT1161), for which a cross-reaction with AT-III was confirmed at about 0.1%, was placed in an EIA tube at 10%.
After immobilization at a concentration of μg / mL, phosphate buffered saline (PBS, pH 7.0) containing 1% w / v bovine serum albumin.
An anti-TAT antibody-fixed tube blocked in 2) was prepared. As a labeled antibody, a sandwich EIA using a commercially available kit of horseradish-derived peroxidase-labeled anti-human AT-III antibody (POD-labeled anti-AT-III antibody) was used.
TAT was measured by the method. The measurement sample was human AT-II
And AT prepared by mixing I and human thrombin
A sample to which -III was added at a concentration of 200 µg / mL was used. In the plasma of normal DIC (general intravascular coagulation) patients, even if TAT is formed, excess AT-II
Because of the remaining I, this artificially prepared TAT
And AT-III can be considered as a replacement for DIC patient plasma.

All the measurement procedures were performed according to the instruction manual of the commercially available kit, and the measurement equipment was Elgia F-300.
Using (International Reagents Co., Ltd.), the signal obtained by the measurement was determined as a relative fluorescence intensity. That is, the measurement sample and the reaction buffer were dispensed into a solid-phase tube and reacted at room temperature for 9 minutes. Then, the unreacted substance was removed by suction, washed, and then a POD-labeled anti-AT-III antibody solution was added and reacted for 9 minutes. Let it. Thereafter, the POD-labeled anti-AT-III antibody solution was removed by suction and washed. After the substrate solution was dispensed and reacted for 9 minutes, the enzyme reaction stop solution was dispensed and the fluorescence intensity was measured.

On the other hand, in order to confirm the effects of the present invention, the same operation as in the above method was carried out using a reagent obtained by adding 10 mM dithiothreitol (DTT) to a reaction buffer and a POD-labeled anti-AT-III antibody solution, respectively. Then, TAT was measured. The results are shown in Table 1.

[0020]

[Table 1] TAT measurement results

As a result, in the case of the reagent without addition of 10 mM DTT, the presence of AT-III
No TAT concentration-dependent TAT-specific signal was obtained due to the signal due to cross-reaction with III.
In the method of the present invention to which DTT was added, even in a sample in which AT-III was present, the TAT concentration-dependent TAT
A specific signal was obtained, indicating that TAT measurement was possible.

[0022]

Example 2 A monoclonal antibody against another TAT having a cross-reactivity of about 0.3% between the monoclonal antibody against TAT used in Example 1 and AT-III (clone N
o. TAT1812) was mixed in equal amounts to give 0.3
Adsorbed on 5 μm polystyrene latex particles,
/ V% bovine serum albumin (BSA) was dispersed in a PBS buffer (pH 7.2), and the concentration of latex particles was adjusted so that the absorbance at a wavelength of 660 nm was about 2.5.

1% w / v in PBS buffer containing BSA
% Of polyethylene glycol 6000, and two kinds of reaction buffers, one with and without 20 mM DTT. Example 1
The sample containing only TAT and the sample obtained by adding AT-III to TAT were measured. The measurement was performed by reacting 20 μL of a sample with 150 μL of a reaction buffer at 37 ° C. for 5 minutes using a Coagrex 800 type coagulation automatic analyzer.
150 μL of a latex reagent was added, and the change in absorbance for 5 minutes was measured. The results are shown in Table 2.

[0024]

Table 2 Effect of DTT on TAT measurement using TAT latex reagent

From the above results, in the case where DTT was not added, in the case of AT-III, if AT-III was present in the sample, no TAT concentration-dependent signal was obtained, and AT-III was not obtained.
A high absorbance change was observed due to coagulation. On the other hand, when DTT was added, a change in absorbance depending on the TAT concentration was required for all samples, and it was found that TAT could be measured specifically.

[0026]

Industrial Applicability The present invention relates to a method for immunologically measuring a test substance, a method for specifically measuring the test substance by measuring in the presence of a substance that reduces cross-reactivity, and a measuring reagent. I will provide a. By introducing a means for reducing the reactivity of contaminating cross-reactive substances, specifically by allowing a substance that reduces the reactivity of contaminating cross-reactive substances to be present in the immune reaction system, the purpose and the purpose which were not possible before were The test substance to be measured can be measured immunologically.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/543 583 G01N 33/543 583 (72) Inventor Masahiro Okuda 1-chome, 1-Muroya, Nishi-ku, Kobe-shi, Hyogo Prefecture. 2 R & D Center in International Reagents Co., Ltd. (72) Inventor Hisahide Hibura 1-2-1-2 Muroya, Nishi-ku, Kobe City, Hyogo Pref.

Claims (7)

[Claims] 1. An immunoassay method for immunologically measuring a test substance, which comprises introducing means for reducing the reactivity of contaminating cross-reactive substances. 2. The immunoassay according to claim 1, wherein the reactivity is an immunological reaction. 3. The immunoassay according to claim 1, wherein the test substance is a thrombin-thrombin inhibitor complex, and the contaminating cross-reacting substance is a thrombin inhibitor and / or prothrombin. 4. The immunoassay according to claim 1, wherein the means for reducing the reactivity is the presence of a substance for reducing the reactivity of contaminating cross-reactive substances. 5. The immunoassay according to claim 4, wherein the substance that reduces the reactivity is a substance that cleaves a disulfide bond of a contaminating cross-reactive substance. 6. A substance that reduces the reactivity of contaminating cross-reactive substances is at least a surfactant, dithiothreitol, mercaptoethylamine, glutathione, mercaptoethanol, monothiophosphoric acid, sodium borohydride, dithioerythritol, thioglycolic acid. Or one or more selected from dithionite, tris (2-carboxyethyl) phosphine, sulfite, α-thioglycerol, and 2-aminoethylisothiouronium hydrobromide. The immunoassay according to claim 4. 7. A measuring reagent kit used in the immunoassay according to claim 1.