JPH06289022A - Standard liquid for immunoassay, buffer liquid for diluting specimen, antibody liquid, and immunoassay method using them - Google Patents

Abstract

PURPOSE:To achieve an accurate immunoassay regardless of storage conditions and incubation conditions by containing a protease inhibitor in the standard liquid for immunoassay, the buffer liquid for diluting specimen, and an antibody liquid. CONSTITUTION:Standard liquid is a liquid which is diluted to various kinds of concentration containing antigen and is used to create a calibration curve. The dilution liquid of a liquid containing a protease such as human colostrum is used as the standard liquid and is especially effective where cancer-related human galactose transferase, etc., are antigens. The examples of the buffer for diluting specimen are varilus kinds of humors such as blood, serum urine, and lymph globe. A liquid where an antibody is dissolved, for example, a second antibody liquid used for the sandwich method, is used for the antibody liquid. Protease inhibitor, etc., are contained in the standard liquid, the buffer liquid for diluting antibody, and the antibody liquid. For example, N-ethylmaleimide, ethylenediaminetetraacetic acid, and its salt, and phennylmethanesulfonyl fluoride are used as the protease inhibitor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a standard solution used for immunoassay, a sample dilution buffer solution and an antibody solution, and an immunoassay method using these.

[0002]

2. Description of the Related Art Conventionally, an immunoassay method has been widely used as a method for measuring a small amount of antigen in the diagnosis of diseases, food inspection and the like. Although various immunoassays have been performed, an antigen standard solution for preparing a calibration curve, a buffer solution for diluting a sample, and an antibody solution in which an antibody used for immunoassay is dissolved may be used. Many. For example, in the immunoassay for cancer-related human galactosyltransferase useful for diagnosing cancer, a diluted human colostrum containing a large amount of cancer-related human galactosyltransferase is used as a standard solution. However, the present inventors have found that when this standard solution is used, the results of immunoassay vary depending on the storage conditions. It was also found that even if the same storage conditions were used, the measurement results varied depending on the incubation conditions, which hindered accurate immunoassay.

[0003]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide means capable of performing an accurate immunoassay regardless of storage conditions and incubation conditions.

[0004]

As a result of earnest research, the inventors of the present invention have found that a proteolytic enzyme is present in a sample, an antigen standard solution or an antibody solution, and the proteolytic enzyme causes an antigen or an antibody to immunoreact. It was found that it is decomposed during the incubation for carrying out the procedure or during storage of the solution, which hinders accurate immunoassay, and that this problem can be solved by including a protease inhibitor in these solutions. And completed the present invention.

That is, the present invention provides a standard solution for immunoassay containing a protease inhibitor. The present invention also provides a sample-diluting buffer for use in immunoassay, which contains a protease inhibitor. Furthermore, the present invention provides an antibody solution for use in immunoassay, which contains a protease inhibitor. Furthermore, the present invention provides an immunoassay method for detecting or quantifying an antigen using at least one of the above-mentioned solutions of the present invention.

The present invention will be described in detail below.

The standard solution in the present invention is a solution obtained by diluting an antigen to various concentrations and is used for preparing a calibration curve. The antigen is not particularly limited and may be any one. It is particularly effective when a cancer-related human galactosyltransferase or the like is used as an antigen, which uses a diluted solution containing a proteolytic enzyme as a standard solution, such as human colostrum, but is not limited thereto.

The sample-diluting buffer solution in the present invention is a buffer solution used for diluting a sample, and its kind is not limited at all. Further, the sample is not limited at all, and various body fluids such as blood, serum, urine, saliva, and lymphocyte fluid can be exemplified, but the sample is not limited thereto.

The antibody solution in the present invention is a solution in which an antibody used for immunoassay is dissolved. For example, a solution of a second antibody, particularly E, used in the sandwich method.
Examples of the enzyme-labeled antibody solution used for LISA include, but are not limited to.

As described above, the standard solution, the sample dilution buffer solution and the antibody solution of the present invention contain a protease inhibitor. Preferred examples of the protease inhibitor include N-ethylmaleimide (hereinafter sometimes referred to as “NEM”), ethylenediaminetetraacetic acid (hereinafter,
Sometimes referred to as "EDTA") and salts thereof, and phenylmethanesulfonyl fluoride. As the salt of EDTA, an alkali metal salt such as sodium salt is preferable. The protease inhibitors may be used in combination of two or more kinds.

The concentration of the proteolytic enzyme inhibitor in these liquids is within a range that can substantially reduce or prevent the decomposition of the antigen or antibody by the proteolytic enzyme and does not interfere with the original immunoassay. If there is, it is not particularly limited. Usually, it is preferably 1 mM to 50 mM, more preferably 2 mM to 20 mM.

In the immunoassay method of the present invention, at least one of the standard solution of the present invention, the sample dilution buffer solution and the antibody solution described above is used. The immunoassay method itself is not particularly limited and may be any conventionally known immunoassay method such as a sandwich method, an agglutination method, or a competition method. The measurement target is not limited at all. It is particularly effective when a diluent such as human whey containing a proteolytic enzyme is used as a standard solution, and a cancer-related human galactosyltransferase, etc. is a measurement target, but is not limited to this. .

[0013]

INDUSTRIAL APPLICABILITY According to the present invention, degradation of an antigen or antibody by proteolytic enzymes during storage and incubation is reduced or prevented, and accurate immunoassay can be performed regardless of storage conditions or incubation conditions. It was

[0014]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Preparation of a standard solution containing a protease inhibitor Human colostrum containing cancer-related galactosyltransferase (GAT) in a very high concentration was diluted to 1/50 with horse serum (GAT concentration 0).
A standard solution was prepared by diluting it to 0 or 1/100.

The proteolytic enzyme inhibitors shown in Table 1 below were added to the above standard solutions at the final concentrations shown in the same table, and stored at 20 ° C or -40 ° C for 3 days. Further, as a control, the one to which the protease inhibitor was not added was also stored in the same manner.

[0017]

[Table 1]

Preparation of antibody-bound beads Mouse monoclonal antibody against cancer-related human galactosyltransferase obtained by the method described in JP-A-3-259093 was dissolved in 0.1M sodium chloride to obtain 0.1M.
Polystyrene beads were dissolved in a carbonate buffer solution (PH 9.15) to a concentration of 10 μg / ml, and surface-washed by ultrasonic treatment in ethanol (Sekisui Chemical's #
80) was immersed and the container was slowly rotated at 4 ° C. to immobilize the antibody overnight. After washing with phosphate buffered saline (hereinafter, PBS), it was immersed in a 1% bovine serum albumin (BSA) -PBS solution at 37 ° C. for a whole day and night. The solution was cut well, frozen at −40 ° C., freeze-dried, and stored in a refrigerator.

Preparation of Enzyme-Labeled Antibody Solution An anti-GAT mouse monoclonal antibody (described in Japanese Patent Laid-Open No. 3-259093) that recognizes a site different from the immobilized antibody was edited by Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, “Enzyme Immunoassay (No. (3rd edition)
It was labeled with peroxidase by the method described in "Igaku Shoin 1987" P108. 1M sodium chloride, 1% BSA, 0.
05% p-hydroxybenzoic acid and this peroxidase-labeled anti-GAT monoclonal antibody were dissolved in a 20 mM phosphate buffer (pH 7.2) to prepare an enzyme-labeled antibody solution.

Immunoassay 50 g of the GAT standard solution prepared in Example 1, 1 M sodium chloride and Tween 20 were used to prepare the antibody-bound beads.
20 mM phosphate buffer containing 0.01% (pH 6.
5) Add to a mixed solution with 200 μl and
Incubated for hours. After washing with PBS, 250 μl of the above enzyme-labeled antibody solution was added, and the mixture was incubated at room temperature for 1 hour. After washing with PBS, 0.3 ml of citrate-phosphate buffer (pH 5.0) containing 0.02% hydrogen peroxide and 3 mg / ml of o-phenylenediamine was added, and color was developed for 30 minutes at room temperature. Then, the color reaction was stopped with 1 ml of 1N sulfuric acid, and the absorbance at 492 nm was measured. Table 2 below shows the average values and the coefficient of variation (C.V.) when the standard solution was repeatedly measured 5 times.

[0021]

[Table 2]

When stored at 20 ° C. for 3 days without adding anything as described above, the absorbance is about 20% as compared with −40 ° C.
It is falling. Also, in the case of storing at −40 ° C. without adding anything, a decrease in absorbance is observed as compared with the case of adding the reagent. Since no increase in absorbance was observed in horse serum, it is considered that nonspecific adsorption did not increase due to the influence of reagents. For CV, if nothing was added, 20 ° C
It clearly deteriorates in storage, and the deterioration tends to occur even at -40 ° C, but the CV of the reagent added is good regardless of the temperature. Therefore, the addition of the reagent inhibited the activity of the proteolytic enzyme during storage and incubation, and as a result, the decrease in absorbance and the deterioration in simultaneous reproducibility were prevented.

Example 2 Preparation of a buffer solution to which a protease inhibitor was added A reagent was added to a 20 mM phosphate buffer solution (pH 6.5) in which 1 M sodium chloride was dissolved so that the final concentration shown in Table 3 below was obtained. The added buffer solutions were prepared respectively.

[0024]

[Table 3]

Immunoassay An immunoassay similar to that of Example 1 was carried out using the above-mentioned buffer solution and the standard solution prepared without adding the reagent prepared in Example 1. Table 4 shows the average values and the coefficient of variation (CV) when the standard solution was repeatedly measured 5 times.

[0026]

[Table 4]

As a result of inhibiting the activity of the proteolytic enzyme during the incubation for carrying out the immunoreaction by adding the reagent, the decrease of the absorbance and the deterioration of the simultaneous reproducibility were prevented.

Example 3 Preparation of Enzyme-Labeled Antibody Solution Added with Proteolytic Enzyme Inhibitor
Preparation of peroxidase-labeled anti-GAT monoclonal antibody and 1
1% BSA-PBA solution containing M sodium chloride (pH
The enzyme-labeled antibody liquids were prepared by adding reagents to the final concentration shown in Table 5 below in 7.2).

[0029]

[Table 5]

Immunoassay Table 6 shows the results of immunoassays similar to those of Example 1 using the above enzyme-labeled antibody solution and the standard solution prepared in Example 1 to which the reagent was not added.

[0031]

[Table 6] The addition of reagents inhibited the activity of proteolytic enzymes during the incubation to drive the immune reaction.

Claims (6)

[Claims] 1. A standard solution for immunoassay containing a protease inhibitor. 2. A sample diluting buffer used for immunoassay, which contains a protease inhibitor. 3. An antibody solution used for immunoassay, which contains a protease inhibitor. 4. The proteolytic enzyme inhibitor is at least one selected from N-ethylmaleimide, ethylenediaminetetraacetic acid and salts thereof, and phenylmethanesulfonyl fluoride, according to any one of claims 1 to 3. Liquid. 5. An immunoassay method for detecting or quantifying an antigen using at least one of the liquids according to claim 1. 6. The immunoassay method according to claim 5, wherein the antigen is a cancer-related human-derived galactosyltransferase.