JPS6246263A - Method for quantifying prostate specific antigen and alpha1-antitrypsin composite - Google Patents

Abstract

PURPOSE:To make it possible to calculate the quantity of a gamma-Sm-a1-antitrypsin composite (gamma-Sm-a1AT) by using an anti-gamma-seminoprotein (gamma-Sm) antibody and an anti-a1-antitrypsin antibody. CONSTITUTION:The titled method can be performed by a sandwich immunoassay method. For example, anti-gamma-Sm antibody is adsorbed by or bonded to an insoluble carrier to form an insolubilized antibody. An anti-a1-antitrypsin antibody is labelled with a marker such as enzyme, an radioactive isotope or a fluorescent substance to obtain a labelled antibody. These antibodies are subjected to the antigen-antibody reaction with the gamma-Sm-a1AT composite in a specimen to be examined and the quantity of said composite can be calculated from the quantity of the bonded marker. Contrarily, the anti-a1-antitrypsin antibody may be converted to an insolubilized antibody while the anti-gamma-Sm antibody may be used as a marked antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION γ-Seminoprotein (γ-Seminopro
tein) (hereinafter abbreviated as γ-SmJ) and αi-antitrypsin (hereinafter referred to as γ-Sm-αI
This invention relates to a method for determining the composition of AT complex (hereinafter abbreviated as "AT complex").

The present inventor, for the purpose of investigating the properties of γ-Sm in serum,
Prostate cancer patient serum was gel-filtered and γ-Sm was detected using the γ-Sm oxygen immunoassay (EIA) method. As a result, low molecular weight (molecular weight approximately 30,000) and high molecular weight 1 (molecular weight approximately 90,000) were detected. γ-Sm was detected.

Low molecular weight γ-Sm is the same as γ-Sm isolated from seminal plasma (molecular weight 33,000-34,000), but it is suggested that high molecular weight γ-Sm is a complex bound to proteins present in serum. It was done. Therefore, as a result of examining proteins present in serum that form complexes with γ-Sm,
It was found that αi-antitrypsin forms a complex with γ-Sm. That is, γ-Sm exists in serum in the form of free γ-Sm and γ-Sm-αIATI combination.

The total amount of free γ-Sm and γ-Sm-αIAT complex can be measured by, for example, a radioimmunoassay (RIA) method using an anti-γ-Sm antibody, an EIA method, or the like. This method is described in the literature 1-day Urology Journal, vol. 74,
pp. 1320-1325, 1983 (Tsutomu Okabe et al.), Clinical Examination, Vol. 28, pp. 1755-1758, 1984 (
It has been reported in Makoto Kamachi et al.

However, γ-3 which measures only the γ-3m-αIAT complex
An immunoassay method using m antibody and anti-αi-antitrypsin antibody was established.

That is, the present invention provides γ-3m and α
i-antitrypone complex (γ-Sm-α+ATi
The present invention provides a method for measuring γ-Sm-αtATm combination and a composition for measuring γ-Sm-αtATm combination containing at least the two types of antibodies.

The present invention is a labeled antibody by labeling an anti-αi-antitrypsin antibody with a 5"H reagent such as an enzyme, a radioisotope, or a fluorescent substance, which is carried out by a sandwich-type immunoassay method commonly practiced in this field. , these two antibodies are allowed to react with the γ-3m-α (AT complex) in the test sample and the antigen-antibody reaction, and the amount of the complex can be determined from the amount of bound labeling agent. Alternatively, the anti-αi-antitrypsin antibody may be used as an insolubilized antibody, while the anti-γ-3m antibody may be used as a labeled antibody.

The anti-γ-Sm antibody and anti-αi-antitrypsin antibody in the present invention may be either polyclonal antibodies or monoclonal antibodies, and each uses γ-3m or αi-antitrib/n as an antigen in a host animal.
Mouse, rat, rabbit.

goat, etc.) and obtain it as a polyclonal antibody from the serum of the animal, or obtain it as a monoclonal antibody from a fused cell of pancreatic cells and myeloma cells of the host animal, either by techniques known in the art. Obtainable.

The insolubilizing carrier used to produce the insolubilized antibody may be a general immunoassay carrier (it may be of any type. The shape of the carrier may be a ball shape, a plate shape, or a Ia shape.

A carrier used for gel filtration, which may be angular or other arbitrary shape;
For example, Sepharose 4B or the like may be used. The material of the carrier may be polystyrene, polyacetate, polycarbonate, plastino resin such as acrylonitrile-butanoene-styrene copolymer, paper such as cellulose, or any other material conventionally used for immunotherapy. . Furthermore, binding of the antibody to the carrier can be achieved by known techniques such as physicochemical absorption or chemical binding.

The labeled antibody is 3H914c, l! 5■,
Radioactive isotopes such as 131 ■, enzymes such as verokindase, alkaline phosphatase, β-galacto/dase, etc.

Fluorescent substances such as fluorenocein and rhodamine B.

It can be obtained by labeling with a labeling agent commonly used in the field, such as a luminescent substance such as an isoluminol derivative, by a conventional method.

Next, a method of carrying out the present invention using an insolubilized antibody and a labeled antibody will be briefly explained.

First, a certain amount of the sample to be measured is placed in a reaction container, and 0.1 to 5% of BSA, unγ-globulin, or animal serum is added to the reaction vessel, preferably 0.5 to 2% of BSA.
Add a certain amount of a buffer containing SA and mix. The buffer in this case is a 0.01-0.5M borate buffer (pH
7,2-9.0), O,Of~0.5M phosphate buffer (pH 5,2-8.5), 0.01-0.5M veronal buffer (pH 6,8-9. 6) or 0.01
~0.5M Tris-HCl buffer (pH 7,2-9.
A buffer such as 1) or the above buffer containing a salt such as 0.05 to 0.5 M sodium chloride or 0.05 to 0.5 M potassium chloride is used. Next, an insolubilized antibody (a carrier bound to an anti-γ-Sm antibody or an anti-αi-antitrypsin antibody) is added to the sample and buffer mixture, and the mixture is allowed to react for a certain period of time. After the reaction, the carrier is optionally washed, and then the labeled antibody (insolubilized antibody is anti-γ-S) dissolved in the above buffer is added.
In the case of the m antibody, use an anti-αi-antitrypsin antibody labeled with a labeling agent, and if the insolubilized antibody is an anti-αi-antitrypsin antibody, use an anti-γ-3m antibody labeled with a labeling agent)
and the carrier for a certain period of time. After the reaction, the carrier is washed and the amount of the labeling agent of the labeled antibody bound to the carrier is detected to determine whether γ-3m-αIAT? You can know the amount of 3J coalescence.

Next, according to a test example, γ-3m in the serum of prostate cancer patients
It will be explained that prostate cancer can be diagnosed by detecting -α7AT complex and measuring only this complex. still,
Anti-γ-S used in test examples and examples described below
m monoclonal antibodies in literature, clinical examination, vol. 28, 17
55-1758, 1984 (Nobu Kamachi et al.) was used.

Test Example 1 Detection of γ-Sm-αiAT complex in the serum of a prostate patient
After loading onto a column (5 x 90 cm), it was eluted with 0.1 M borate buffer (pH 8,0) containing 0.9% sodium chloride, and 10 ml aliquots were collected. 100 pieces each
Using μm, the above-mentioned literature, Clinical Examination, Vol. 28, 175
Anti-γ-Sm monoclonal antibody binding AB according to the measurement procedure of 5-IT'758, 1984 (Shin Kamachi et al.)
Free γ-Sm and γ-Sm-α7AT using S beads and peroxidase-labeled anti-γ-3m antibody? ! [Both the coalescence was measured together, and on the other hand, according to Example (4) described below using anti-γ-Sm monoclonal antibody-bound ABS beads and peroxidase-labeled anti-αi-antitrypsin antibody,
Only the γ-Sm-αIAT complex was measured. Results first
Shown in the figure.

Based on this result, the above-mentioned document, Clinical Examination, Volume 28.

When anti-γ-3m antibodies are used for both insolubilized antibodies and enzyme-labeled antibodies according to Nobu Kamachi et al., 1984, pp. 1755-1758, free γ-3m and γ-3m-α+A
T? Both S1 conjugates were detected, while the insolubilized antibody and the enzyme-labeled antibody were combined with different antibodies (anti-γ-3m antibody and αi
-antitrypsin antibody) by using γ-S
Only the m-αIAT complex can be detected.

Test Example 2 Examination of the relationship between γ-3m-αIAT151 combination in serum and prostate cancer γ-3m-αIAT in serum of 19 normal subjects, 19 patients with benign prostatic hyperplasia, and 19 patients with prostate cancer (untreated) The composite was measured according to Example (4) described below. The results are shown in FIG. As is clear from the results, the concentration of γ-Sm-αIAT complex in the serum of prostate cancer patients is higher than that of normal subjects and subjects with benign prostatic hyperplasia! It was once. As is clear from the above test examples, the γ-Sm-αLA of the present invention
Measurement of T complex is useful in the diagnosis of prostate cancer.

Next, the present invention will be explained by examples.

Example (1) Preparation of insolubilized antibody Anti-γ-Sm monoclonal antibody or anti-αi-antitrypsin antibody was adjusted to 15 μg/ml in 0.1 M borate buffer (pH 8.0) containing 6% sodium chloride.

Wash spherical (6.3 S mm diameter) acrylonitrile-butadiene-styrene (ABS) beads thoroughly with detergent and distilled water, and add 10 ABS beads to 20 ml of each antibody solution.
0 pieces were soaked. After standing at 37°C for 24 hours, the ABS beads were washed with 0.9% sodium chloride solution, 1% town clear albumin, 0.1% sodium azide and 0.9% sodium chloride solution.
0.1 M borate buffer (pH 7) containing sodium chloride
, 4) and stored at 4°C.

(2) Preparation of enzyme-labeled antibody 1) Peroxidase-labeled anti-γ-3m antibody Anti-γ-Sm FRM of F(ab')2 from anti-γ-Sm rabbit serum by a conventional method,
-Reduction with mercaptoethylamine/hydrochloric acid to obtain Fab'. Next, a maleimide group was introduced into peroxidase using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide, and F
A peroxidase-labeled anti-γ-Sm antibody was prepared by reacting with the thiol group of ab'.

+1) peroxidase-labeled anti-αI-antitrypton antibody peroxidase-labeled anti-αi-antitrypton antibody (
Goat (manufactured by Kappel) was diluted to an appropriate amount. ' (3) Preparation of 7-S m -αIATi combination 2 ml of r-Sm solution (2.6 mg/ml) in 6 ml of human serum
was added and left at 37°C for 48 hours, the mixture was added to a Sephadex G-100 column (5 x 90 cm) and 0.
．． 0.1 M borate buffer containing 9% sodium chloride, overnight (
It was eluted at pH 8.0) and fractionated into Sml portions. γ-Sm
- The fractions containing the αIA Ti complex were collected and applied to a Sepharose 4B column bound with an anti-γ-Sm monoclonal antibody (
IXlocm). After washing the column with approximately 50 ml of 0.1 M borate buffer (pH 8,0) containing 0.5% sodium chloride, it was washed with approximately 50 ml of 0.1 M borate buffer (pH 8,0) containing 3.5 M potassium thousandocyanate. 0),
Aliquots of 3 ml were taken. Fractions containing the γ-Sm-αIAT complex were collected, IQ approximately Sm1), and 0.1M borate buffer containing 0.9% sodium chloride (pH 8,0
) 51. After dialysis, Cefazo, Cus G-
100 column (2% 90 cm) containing 0.9% chloride).・1M borate buffer (pH
8,0) and aliquots of 3 ml were collected.

γ-3m-αiAT? The γ-Sm-α, AT complex was obtained by collecting and concentrating the fractions containing 1 complex.

In addition, the absorbance at 280 nm was measured, and the absorbance i,
The concentration showing ooo is 1 unit/ml γ-Sm-α, A
It was defined as T1 coalescence. Based on this, γ-Sm-αIAT? in Example (4)? A standard solution of one combination was prepared.

(4) Measurement of γ-Sm-αIAT complex Transfer 100 μl of the sample or standard solution to a test tube and add 200 μl of 0.1 M borate buffer (pH 7.4) containing 1% bovine serum albumin and 0.3 M sodium chloride. was added and stirred. One anti-γ-Sm monoclonal antibody-conjugated ABS bead was added to this, and the mixture was left at 4°C for 15 hours. 0.05% Tween 2
The ABS beads were washed twice with 4 ml of physiological saline containing 0.

Add 300 μm of peroxidase ff1m anti-αi-antitrypsin antibody solution (prepared by diluting with 0.1 M borate buffer containing 1% bovine serum albumin and 0.3 M sodium chloride, pH 7.4), and incubate at room temperature for 2 hours. I left it for a while. 0
．． ABS with 4 ml of physiological saline containing 05% Tween 20
Beads were washed. Citric acid-phosphate buffer (pH 6,0) containing 0.2% 0-phenyleneamine dihydrochloride and 0.015% hydrogen peroxide in a new test tube, Sml
Put the washed ABS beads into it and let it cool for 1 hour at room temperature.
I left it for a while. Add 2.0 ml of 2N sulfuric acid and stir.
The absorbance at 92 nm was measured, and 78 degrees of the γ-Sm-αIATff1 combination in the sample was determined from the standard curve.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram showing separation of free γ-3m and γ-Sm-αiA T 1 combination in prostate cancer patient surface 7N in Test Example 1. The solid line shows the results of detection using an anti-γ-Sm monoclonal antibody as the insolubilized bt body and an anti-αi-antitrypsin antibody as the enzyme-labeled antibody. The dashed line indicates anti-γ-
The results of detection using the 3m antibody are shown. Figure 2 shows γ-Sm-αIATi in the serum of normal subjects, benign prostatic hyperplasia (BPH) patients, and prostate cancer (PCa) patients.
This is a graph showing the combined 14 degrees (μUnit/ra. Off diagram! S θ l-0/10 10 J?D
/ρO test, 6' with Shun;? To the talent map

Claims (4)

[Claims] (1) A method for quantifying a γ-Sm-α_i-antitrypsin complex, characterized by using an anti-γ-Sm antibody and an anti-α_i-antitrypsin antibody. (2) The method according to claim 1, wherein either the anti-γ-Sm antibody or the anti-α_i-antitrypsin antibody is insolubilized, and the other is a labeled antibody. (3) γ-S comprising at least two anti-γ-Sm antibodies and anti-α_i-antitrypsin antibodies
A reagent for quantifying m-α_i-antitrypsin complex. (4) The quantitative reagent according to claim 3, wherein either the anti-γ-Sm antibody or the anti-α_i-antitrypsin antibody is insolubilized and the other is a labeled antibody.