JP4390963B2 - Immunoassay method and reagent kit - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention is used in an immunoassay method and a reagent kit for measurement of a test substance in a biological sample in the clinical laboratory field.
[0002]
[Prior art]
Measurement of a test substance in a sample using an immunological reaction is routinely performed. The measurement is performed on various measurement principles depending on the concentration of the test substance. Among them, immunoturbidimetry, latex turbidimetry, enzyme immunoassay and the like are typical methods. The antibody used for the immunological reaction is prepared using a test substance as an antigen, and generally exhibits a cross-reaction with a similar substance of the antigen. Therefore, when a test substance and its similar substance, that is, a cross-reactive substance coexist in a sample, an accurate measurement result cannot be obtained if the antibody used for immunological measurement has a high cross-reactivity. Therefore, in general, an antibody having low cross-reactivity is selected and an appropriate measurement system is constructed.
[0003]
When measuring the concentration of thrombin and antithrombin-III complex (TAT), a sandwich enzyme immunoassay method using antibodies specific for each of thrombin and antithrombin-III (AT-III) constituting the complex. Methods for measuring are known. If this TAT is to be measured by latex immunoturbidimetry, it is necessary to prepare an antibody that is specific for TAT and does not cross-react with thrombin, prothrombin and AT-III. However, while the TAT concentration in plasma is usually about 1 to 50 ng / mL, the concentration of AT-III is about 250 μg / mL, which is 5,000 to 250,000 times higher. Therefore, it is necessary to prepare a monoclonal antibody that is TAT-specific and has a cross-reactivity with AT-III of 1 / 50,000 or less, but it is difficult to prepare such a monoclonal antibody. The reagent for measuring TAT by is not put into practical use.
[0004]
On the other hand, when the antibody titer against a pathogen or the like produced in a biological sample is measured by an immunoaggregation reaction such as hemagglutination, the sample is subjected to dithiothrease in order to examine whether the antibody is IgG type or IgM type. A method is known in which the antibody activity of an IgM type antibody is reduced by treatment with a reducing agent such as Toll, and the presence of the IgM type is confirmed from the degree of reduction in aggregation obtained. However, in the method for measuring an antigen, it is unthinkable to specifically measure a test substance by chemically treating contaminating cross-reacting substances.
[0005]
[Problems to be solved by the invention]
The present invention, thrombin - a method for measuring antithrombin III complex immunologically, even coexist antithrombin III and prothrombin cross-reactive substances in the sample, the thrombin - antithrombin III specific complexes It is providing the method and measuring reagent which measure automatically.
[0006]
[Means for Solving the Problems]
The present inventors have results of extensive studies, by the presence of a quality material to cleave the disulfide bond cross-reacting substances contaminating the sample in the immune response system, which was conventionally impossible, thrombin of interest - The inventors have found that the antithrombin III complex can be measured immunologically and have completed the present invention.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The present invention introduces a means for reducing the reactivity of contaminating cross-reactive substances in a method for immunologically measuring a test substance, and more specifically, the presence of a substance that reduces the reactivity of contaminating cross-reactive substances. It is an immunoassay method characterized by this.
[0008]
In the present invention, as a substance that can reduce cross-reactivity, a substance or a surfactant that cleaves a disulfide bond is effective. Specifically, dithiothreitol, mercaptoethylamine, glutathione, mercaptoethanol, monothiophosphoric acid, sodium borohydride, dithioerythritol, thioglycolic acid, dithionite, tris (2-carboxyethyl) phosphine, sulfite, In addition to substances that cleave disulfide bonds such as α-thioglycerol and 2-aminoethylisothiouronium hydrobromide bromide, examples include acids, alkalis, chaotropic compounds, water-soluble organic solvents, surfactants, and the like. It is not limited to these. Further, these disulfide bond cleavage substances may be stabilized using a chelating agent such as EDTA. These substances can be used alone or in combination of two or more. The concentration to be used can be easily determined by using an antibody used for measurement of the test substance and comparing the cross reaction between the test substance and the cross-reacting substance in the presence of these substances.
[0009]
Further, as described above, the means for reducing the reactivity of the contaminating cross-reactive substance is preferably achieved by adding a specific substance. Based on knowledge of immunological reactivity, for example, the ionic strength of the reaction solution can be reduced. It is also possible to adjust the pH, adjust the reaction temperature, etc. That is, if the cross-reacting 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. Through repeated experiments, the present invention can be easily applied to the present invention using the level of specificity of the substance as an index.
[0010]
Substances that can be measured by the immunoassay method of the present invention include substances that cannot be measured accurately due to cross-reactive substances contaminated in the sample, such as thrombin-thrombin inhibitor complex, plasmin-plasmin inhibitor complex, thrombin, and the like. Hirudin complex, elastase and α1-antitrypsin complex, trypsin and trypsin inhibitor complex, active factor X and AT-III complex, protein C and protein C inhibitor complex, tissue plasminogen activator and plasminogen activator inhibitor complex Active 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 or Rotein G and immunoglobulin complex conjugates and the like.
[0011]
In addition, the test substance is not limited to these, and can be used for any substance in which a cross-reactive substance coexists in the sample in the immunoassay method of the test substance. That is, the immunoassay method of the present invention is useful for the measurement of these complexes, and in the case where a substance that is originally intended for measurement cannot be accurately measured by a cross reaction, a substance that can reduce the cross reaction coexists. Applicable to substances that can adopt the method.
[0012]
Specifically, a thrombin-thrombin inhibitor complex is preferably exemplified as the test substance. In this case, the contaminating cross-reacting substance is a thrombin inhibitor and / or prothrombin. A typical example of a thrombin inhibitor is AT-III.
[0013]
As described in detail in the following examples, a latex reagent in which a monoclonal antibody having a cross-reactivity with AT-III of about 1/1000 is adsorbed on latex particles is prepared, and the prepared latex reagent and plasma sample are prepared. Mix and measure the agglomeration process of the latex optically. In the plasma sample, AT-III is present in an amount 5,000 to 250,000 times as large as the test substance TAT. Therefore, this reagent also reacts with AT-III. Gives a signal about 5 times higher than the upper limit (50 ng / mL) (250 ng / mL). 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-reacting substance of the present invention, a measured value of 2.5 ng / mL or less is obtained. Obtained and clinically useful test measurement method can be provided.
[0014]
In the examples, dithiothreitol (DTT) is used as an example of a substance that reduces the reactivity of AT-III in the measurement of the thrombin-thrombin inhibitor complex. Of course, the above substances should also be used as appropriate. Can do.
[0015]
Examples of immunological measurement methods to which the present invention can be applied include known enzyme immunoassay methods (EIA), turbidimetric methods using carriers or immunoturbidimetric methods using no carriers, immunochromatographic methods, hemagglutination, etc. An immunoassay is mentioned.
[0016]
Furthermore, the present invention provides a measurement reagent kit used in the above immunoassay method.
[0017]
【Example】
[Example 1]
After immobilizing a monoclonal antibody (clone NO.TAT1161) against TAT, which has been confirmed to have about 0.1% cross-reactivity with AT-III, in an EIA tube at a concentration of 10 μg / mL, 1 w / v% cow An anti-TAT antibody-fixed tube blocked with phosphate buffered saline (PBS, pH 7.2) containing serum albumin was prepared. As the labeled antibody, TAT was measured by a sandwich EIA method using a horseradish-derived peroxidase-labeled anti-human AT-III antibody (POD-labeled anti-AT-III antibody). As a measurement sample, a sample to which TAT and AT-III prepared by mixing human AT-III and human thrombin were added at a concentration of 200 μg / mL was used. In the plasma of normal DIC (generalized intravascular coagulation syndrome) patients, excess AT-III remains even if TAT is formed, so this artificially prepared mixture of TAT and AT-III is Can be regarded as a substitute for DIC patient plasma.
[0018]
All the measurement procedures were operated according to the instruction manual of the commercially available kit, the measurement instrument was Elgia F-300 (Kokusai Reagent Co., Ltd.), and the signal obtained by the measurement was determined as relative fluorescence intensity. That is, a measurement sample and a reaction buffer solution are dispensed into a solid phase tube, reacted for 9 minutes at room temperature, unreacted substances are removed by suction, washed, and then a POD-labeled anti-AT-III antibody solution is added and reacted for 9 minutes. Let Thereafter, the POD-labeled anti-AT-III antibody solution was removed by suction and washed, and then the substrate solution was dispensed and reacted for 9 minutes, and then the enzyme reaction stop solution was dispensed to measure the fluorescence intensity.
[0019]
On the other hand, in order to confirm the effect of the present invention, TAT was prepared by the same operation as described above using a reagent obtained by adding 10 mM dithiothreitol (DTT) to the reaction buffer solution and the POD-labeled anti-AT-III antibody solution. It was measured. The results are shown in Table 1.
[0020]
[Table 1]
TAT measurement result ┌───────┬────────────┬────────────┐
│ TAT concentration │ Reagent without DTT │ Reagent with 10 mM DTT │
│ (ng / mL) ├─────┬──────┼─────┬──────┤
│ │ TAT only │ TAT + AT-III │ TAT only │ TAT + AT-III │
├───────┼─────┼──────┼┼─────┼──────┤
│ 0 │ 3.2 │ 350.8 │ 2.9 │ 3.4 │
│ 5 │ 12.3 │ 352.2 │ 13.7 │ 14.1 │
│ 10 │ 22.5 │ 348.3 │ 24.3 │ 25.9 │
│ 20 │ 41.6 │ 351.9 │ 42.1 │ 42.9 │
│ 30 │ 65.3 │ 357.9 │ 66.8 │ 66.1 │
│ 50 │ 124.9 │ 347.5 │ 129.4 │ 128.4 │
└───────┴─────┴──────┴┴─────┴──────┘
[0021]
As a result of the above, if AT-III is present in the sample in the reagent without addition of 10 mM DTT, a TAT concentration-dependent TAT-specific signal cannot be obtained due to a signal due to a cross-reaction with AT-III. In the method of the present invention to which 10 mM DTT was added, it was found that a TAT concentration-dependent TAT-specific signal was obtained even in a sample in which AT-III was present, and TAT could be measured.
[0022]
[Example 2]
Two equivalents of a monoclonal antibody against another TAT (clone No. TAT1812) having about 0.3% cross-reaction between the monoclonal antibody against TAT and AT-III used in Example 1 were mixed in an equal amount. Adsorbed to 35 μm polystyrene latex particles, dispersed in PBS buffer (pH 7.2) containing 1 w / v% bovine serum albumin (BSA), and latex particles so that the absorbance at a wavelength of 660 nm is about 2.5. The concentration of was adjusted.
[0023]
Two types of reaction buffers were prepared, with or without 20 mM DTT added to a latex reaction buffer obtained by adding 1 w / v% polyethylene glycol 6000 to the PBS buffer containing BSA. The TAT-only sample used in Example 1 and the sample in which AT-III was added to TAT were measured. The measurement was carried out by reacting 20 μL of the sample and 150 μL of the reaction buffer solution at 37 ° C. for 5 minutes using a Coagrex 800 coagulation automatic analyzer, and then adding 150 μL of a latex reagent and measuring the amount of change in absorbance for 5 minutes. . The results are shown in Table 2.
[0024]
[Table 2]
Effect of DTT on TAT measurement with TAT latex reagent ┌──────┬────────────┬──────────────┐
│ TAT concentration │ No DTT added (ΔA660 / 5 min) │ 20 mM DTT added (ΔA660 / 5 min)
│ (ng / mL) ├─────┬──────┼──────┬───────┤
│ │ TAT only │ TAT + AT-III │ TAT only │ TAT + AT-III │
├──────┼─────┼──────┼──────┼───────┤
│ 0 │ 0.005 │ 0.412 │ 0.006 │ 0.005 │
│ 5 │ 0.034 │ 0.422 │ 0.029 │ 0.033 │
│ 10 │ 0.072 │ 0.437 │ 0.070 │ 0.070 │
│ 20 │ 0.151 │ 0.429 │ 0.148 │ 0.152 │
│ 30 │ 0.216 │ 0.449 │ 0.223 │ 0.219 │
│ 50 │ 0.321 │ 0.428 │ 0.318 │ 0.327 │
└──────┴─────┴──────┴──────┴───────┘
[0025]
From the above results, when DTT is not added, AT-III does not give a TAT concentration-dependent signal if AT-III is present in the sample, and there is a high absorbance change due to aggregation by AT-III. Observed. On the other hand, when DTT was added, the absorbance change depending on the TAT concentration was determined for any sample, and it was found that TAT can be specifically measured.
[0026]
【The invention's effect】
The present invention, thrombin - a method for measuring antithrombin III complex immunologically, by measuring in the presence of a substance that cleaves the disulfide bond cross-reactants, thrombin - specific antithrombin III complex A measuring method and a measuring reagent are provided. By the quality ones to cleave the disulfide bond cross-reacting substances contaminating present in the immune response system, thrombin and previously impossible in a purpose - antithrombin III complex can be measured immunologically.

Claims (3)

Thrombin - A method for measuring antithrombin III complex immunologically, the immune response for the measurement in the presence of a substance which cleaves the disulfide bonds of antithrombin III及beauty prothrombin cross-reactive substances contaminating An immunoassay method characterized by being performed. Antithrombin III及beauty agent to cleave the disulphide bonds of prothrombin cross-reactive substances contaminating the at least dithiothreitol, mercaptoethylamine, glutathione, mercaptoethanol, phosphorothioate, sodium borohydride, dithioerythritol, thioglycolic One or more selected from acid, dithionite, tris (2-carboxyethyl) phosphine, sulfite, α-thioglycerol, and 2-aminoethylisothiouronium hydrobromide The immunoassay method according to claim 1 . A measurement reagent kit for immunologically measuring a thrombin -antithrombin III complex,
An antibody that binds to a thrombin -antithrombin III complex;
A substance which cleaves the disulfide bonds of antithrombin III及beauty prothrombin cross-reactive substances contaminating,
A reagent kit for measurement comprising