JPH0425763A - Method for determining glutathione s-transferase pi and reagent for diagnosis - Google Patents

Abstract

PURPOSE:To stabilize the content of GST-pi in a specimen over a long period of time by using the supernatant obtd. by subjecting human blood to at least two times of centrifugal sepn. operations as the specimen. CONSTITUTION:The centrifugal sepn. operations are executed at least twice, for 3 to 25 minutes at 300 to 2000Xg more preferably 5 to 20 minutes at 500 to 1600Xg and further for about 20 minutes at about 500Xg and for 10 to 20 minutes at about 1100Xg or for 5 to 10 minutes at about 1600Xg at room temp. or under cooling. The GST-pi in the pretreated specimen is determined by an ordinary immunological method. The content of the GST-pi in the specimen is stabilized over a long period of time in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the field of improved quantitative methods and reagents for clinical diagnosis.

PRIOR ART AND PROBLEMS TO BE SOLVED Glutathione S-transferase (hereinafter referred to as GST) is an enzyme known as an enzyme that catalyzes the conjugation reaction of reduced glutathione with various substances that enter the living body from outside the body or that occur within the living body. There is.

GST is contained in large quantities in the liver, liver, kidneys, placenta, small intestine, pancreas, etc., and is also present in trace amounts in red blood cells, white blood cells, platelets, etc. Acidic GST, neutral GST and basic GST are known as GST isozymes. Among acidic GSTs, those derived from the placenta are GS
It is named T-π.

Detecting the level of GST-π in body fluids such as human serum is said to be useful for diagnosing liver cancer, stomach cancer, etc.
However, it is not known that GST-π levels in human serum can be used as a marker for urinary system cancers such as renal cell carcinoma and urothelial II tumors.

Radioimmunological assay (RIA) and enzyme immunoassay (EIA) have been reported as methods for quantifying GST-π.

By the way, when the present inventors were quantifying serum GSTπ levels in healthy subjects, they often found that while normal quantitative values were obtained in one group, apparently high quantitative values were obtained in another group. . The inventors further investigated that when using human serum as a test sample, blood cells, especially platelets, are destroyed during blood coagulation and acidic GST leaks out, and when using plasma as a test sample, long-term storage at low temperatures is required. Freezing and thawing destroys blood cells, especially platelets, and acidic GST flows out.
We obtained new knowledge that these may be the reasons why quantitative values appear to be high.

As a result of various studies, the present inventors succeeded in removing the apparently abnormally high quantitative values by applying extremely simple pretreatment to human blood.
It was discovered for the first time that the -π level can be used as a diagnostic marker for urinary cancer, and the present invention was completed.

Components of the Invention The first aspect of the present invention is a method for immunologically quantifying GST-π, which is characterized in that a supernatant obtained by centrifuging human blood at least twice is used as a test substance. This relates to a method for quantifying -π.

The centrifugation operation here is performed at least twice.

Centrifugation is carried out at 300-2000 x g for 3-25 minutes, preferably at 500-1600 x g for 5-20 minutes, particularly preferably at about 500 x g for around 20 minutes or about ll0
0X g for 10-20 minutes or about 1600X g for 5~! It is carried out for 0 minutes at room temperature or under cooling. Prior to the centrifugation operation, an anticoagulant such as heparin sodium or EDTA2Na is preferably added to prevent blood coagulation. Thus, GS in the specimen
The T-π content is stabilized over time.

GST-π in the pretreated specimen is quantified by a conventional immunoassay method. As the immunological quantitative method, any method using antigens and/or antibodies labeled with various labels can be applied. Examples of labels include peroxidase, alkaline bosphtase, β-
Examples include enzymes such as galactosidase, radioactive substances such as 1257, and fluorescent substances such as fluorescein inthiocyanate. As an immunological quantitative method,
EIA method where the labeling substance is an enzyme, especially sandwich EI
Method A is listed as a preferred method. Santoifuchi E
In the IA method, antibodies are used as two reagents with different purposes. That is, one of the two is used as an enzyme-labeled antibody conjugated to an enzyme, and the other is used as an insoluble antibody conjugated to an insoluble carrier such as polystyrene beads. One of the two antibodies may be a monoclonal antibody and the other a polyclonal antibody, both may be either a monoclonal antibody or a polyclonal antibody, or they may be 7 lagmates of antibodies. When both are monoclonal antibodies,
The antigenic determinants of each are often different. A combination in which both antibodies are monoclonal antibodies is preferred in terms of uniform quality and continuous supply. Monoclonal antibodies can be produced by Milstein's cell fusion method.

In the German/Deutsch ETA method, an antigen, that is, GST-π, is introduced between the enzyme-labeled antibody and the insoluble antibody to form a Germanic/Deutsch-type antigen-antibody complex, and an enzyme substrate is applied to this to cause an enzyme reaction. The amount of antigen is determined using the enzyme activity as an indicator, and can be carried out according to conventional methods.

A second aspect of the present invention relates to a reagent for diagnosing urinary cancer, which is composed of at least an insoluble anti-GST-π antibody and/or a labeled anti-GST-π antibody.

The reagent of the present invention is used to detect urinary cancer patients and to monitor the progress of treatment and cancer progression by detecting the GST-π level in body fluids such as human serum. As already mentioned, GST-π, which is a marker for liver cancer and gastric cancer, is known, but GST-π, which is a marker for urinary system cancer, is not known.

The reagents of the present invention are substantially the same as those used in the immunoassay method described above. For example, the reagent of the present invention when detecting GST-π levels by the Santoifuchi EIA method is composed of at least: ■ an insoluble anti-GST-π antibody ■ an enzyme-labeled anti-GST-π antibody, and in addition: ■ a substrate for the labeled enzyme ■ a standard GST-π ■ Buffering agent ■ Washing solution ■ Reagents such as enzyme reaction terminators are used as necessary.

If the GST-π level in human blood is higher than that of a healthy person, a urinary system disease such as renal cell carcinoma, upper urinary tract ulcer, bladder ulcer, or prostate cancer is suspected.

Specific Examples Next, the present invention will be explained in more detail by referring to Reference Examples and Examples.

Reference example 1 2 with monoclonal antibody GST-π
．． l of BALB/C mice immunized for 5 months.
ll1! Visceral cells and mouse myeloma cells (XEi3^g
8. lli, 5.3') were subjected to cell fusion according to Milstein's method and cloned to produce two hybridomas A and B that have the following common characteristics but differ in antigenic determinants. was established.

Common characteristics of anti-GST-π antibodies ■ Recognizes GST-π ■ Cross-reacts with some acidic GSTs Reference example 2 that does not cross-react with neutral and basic GSTs
Hybridoma A obtained in Insoluble Antibody Reference Example 1 is cultured in vfvo to obtain the desired anti-GST-π monoclonal antibody A.

Polystyrene beads (Shimizu Chemical) with a diameter (i, 5 mm) were washed with Scato 20X-PF (Nacalai Tesque), purified water was added, and a solution containing 100 mg of monoclonal antibody A was added to the beads. Add 2 at 4℃
Leave it for 4 hours. Polystyrene beads with 0.1% bovine serum albumin-0,1 MIJ:/@buffer (f) H7,0
) to obtain insoluble antibodies.

Reference Example 3 Enzyme-labeled antibody Hybridoma B obtained in Reference Example 1 is cultured in ViVO to obtain the desired anti-GST-π monoclonal antibody B.

40 mg of peroxidase was dissolved in 1.25% glutaraldehyde aqueous solution, and Sephadex G-25 (1.
90 cm). Add an aqueous solution containing 20 mg of monoclonal antibody B and 0.4 rnl of IM charcoal@buffer (pH 9,5) to 6 ml of the peroxidase fraction, and leave it at 4°C for 24 hours [[Sephacryl S-300HR column]. gel filtration to obtain an enzyme active fraction (enzyme-labeled antibody). 0.4ml of this solution
19.6 ml of 0.5% gelatin-0.1% bovine serum albumin-18.79A horse serum-0.01% Tolidor X-4050, 3M NaCI-10mM Phosphorus W1 buffer (pH 7.0) was added. This was used as an enzyme-labeled antibody solution in Example 1 below.

Example 1 S/D EIA standard GST-π
One polystyrene bead (insoluble antibody) obtained in Reference Example 2 and 0.3 ml of the enzyme-labeled antibody solution obtained in Reference Example 3 are added to 50 μl of the solution or test sample, and the mixture is allowed to stand at 37°C. 6
After 0 minutes, the polystyrene beads were soaked in 2 ml of O, 15M N.
After washing twice with aC10,01M phosphate buffer (pH 7,0), transfer the polystyrene beads to another test tube. 0
．． Add 5 ml of enzyme substrate solution [0.3% O-phenylenediamine-0.02% hydrogen peroxide-20mM citrate buffer (pH 6.5>) and let stand at room temperature for 30 minutes, 6N sulfuric acid Stop the reaction by adding 2 ml of
Measure the absorbance in nm.

Figure 1 is a quantitative standard curve.

Example 2 Pretreatment of specimen and determination of GST-π Heparin sodium was added to human blood and stored at 4°C. After 6 hours, it was centrifuged at 1100 x g for 10 minutes at 4°C, and the supernatant was collected again. Centrifuge under the same conditions to obtain a supernatant. The GST-π content of each supernatant that was stored as a solution at room temperature or frozen was determined according to Example 1, and the following results were obtained.

As shown in Table 1, stable quantitative values were obtained by centrifugation twice.

Example 3 Pretreatment of specimen and determination of GST-π Human blood was added with heparin sodium and stored at room temperature, and after 5 hours, centrifuged at 500 x g for 20 minutes at room temperature.
The supernatant is centrifuged again under the same conditions to obtain a supernatant. The GST-π content of each supernatant stored as a solution at room temperature or frozen was determined according to Example 1, and the following results were obtained.

As shown in Table 2, stable quantitative values were obtained by centrifugation twice.

Example 4 GST-π level of patient serum Healthy subjects (8
Blood was collected from 8 cases) and urinary system cancer patients (67 cases), pretreated in the same manner as in Example 2, and the GST-π level was measured in the same manner as in Example 1. The following results were obtained. I got it.

(Hereinafter, blank spaces) From the table in front of Table 3, it can be seen that the GST-π level in serum is a useful marker for urinary system cancer disease.

[Brief explanation of drawings]

FIG. 1 is a quantitative standard curve for the sandwich EIA method.

Claims (4)

[Claims] (1) Glutathione S-transferase π (hereinafter referred to as
1. A method for the immunological determination of GST-π, characterized in that a supernatant obtained by subjecting human blood to at least two centrifugation operations is used as a test substance. (2) The quantitative method according to claim 1, wherein the centrifugation is carried out at 300 to 2000 xg for 3 to 25 minutes. (3) A reagent for diagnosing urinary system cancer comprising at least an insoluble anti-GST-π antibody and/or a labeled anti-GST-π antibody. (4) Insoluble anti-GST-π antibody and enzyme-labeled anti-GST
4. The reagent according to claim 3, which comprises both the -π antibody and a substrate for the labeled enzyme, and both antibodies are monoclonal antibodies having different antigenic determinants.