JPH0690203B2 - Method for quantifying prostate-specific antigen and α-lower 1-antitrypsin complex - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is a protein γ that is specifically present in human seminal plasma.
-Seminoprotein (γ-Seminoprotein)
-Sm ") and α ₁ -antitrypsin complex (hereinafter abbreviated as" γ-Sm-α ₁ AT complex ").

The present inventor, for the purpose of investigating the properties of γ-Sm in serum, gel filtration of prostate cancer patient serum, γ-Sm was detected by enzyme immunoassay (EIA) method of γ-Sm, low molecular weight ( Γ-Sm for fractionation of high molecular weight (molecular weight about 90,000)
Was detected. Low molecular weight γ-Sm is the same as γ-Sm (molecular weight 33000-34000) isolated from seminal plasma, but high molecular weight γ-Sm suggests that it is a complex bound to a protein present in serum. Was done. Therefore, as a result of examining the protein present in serum forming a complex with γ-Sm, α ₁
-It was found that antitrypsin forms a complex with γ-Sm. That is, γ-Sm is free γ in serum.
It exists in the form of -Sm and γ-Sm-α ₁ AT complex.

The total amount of free γ-Sm and γ-Sm-α ₁ AT complex can be measured by, for example, a radioimmunoassay (RIA) method using an anti-γ-Sm antibody or an EIA method. This method is reported in the literature, Journal of the Urology, Vol. 74, 1320-1325, 1983 (Tsutomu Okabe et al.), Laboratory, Vol. 28, pp. 1755-1758, 1984 (Shinichi Kamaike et al.). .

However, it is impossible to measure only the γ-Sm-α ₁ AT complex by the method described in the above literature. The present inventor has developed an anti-γ-Sm antibody and an anti-α for the purpose of measuring only the complex.
An immunoassay method using a ₁ -antitrypsin antibody was established.

That is, the present invention is an anti-gamma-Sm antibodies and anti alpha ₁ - consists of using antitrypsin antibody gamma-Sm and alpha ₁ -
Method for measuring antitrypsin complex (γ-Sm-α ₁ AT complex) and γ-Sm containing at least the two antibodies
It is a composition for -α ₁ AT complex measurement.

The present invention can be carried out by a sandwich-type immunoassay method generally practiced in this field. For example, an anti-γ-Sm antibody is adsorbed or bound to an insoluble carrier to give an insolubilized antibody, and an anti-α ₁ -antitrypsin antibody is also used. A labeled antibody is prepared by labeling with a labeling agent such as an enzyme, a radioisotope or a fluorescent substance, and these two antibodies are allowed to react with the γ-Sm-α ₁ AT complex in the test sample by an antigen-antibody reaction, and the bound label is bound. The amount of the complex can be determined from the amount of the agent. In contrast to the above, anti-α ₁ -antitrypsin antibody may be used as an insolubilized antibody, while anti-γ-Sm antibody may be used as a labeled antibody.

The anti-γ-Sm antibody and the anti-α ₁ -antitrypsin antibody in the present invention may be either polyclonal antibodies or monoclonal antibodies, and each host animal (mouse, rat) using γ-Sm or α ₁ -antitrypsin as an antigen. , Rabbit, goat, etc.) to obtain a polyclonal antibody from the serum of the animal, or a monoclonal antibody from a fused cell of spleen cells and myeloma cells of the host animal. Can be obtained by the technology of.

The insolubilized carrier for producing the insolubilized antibody may be a carrier for general immunoassay, and may be arbitrary. The shape of the carrier may be ball-shaped, plate-shaped, rod-shaped, square-shaped or any other shape, and a carrier used for gel filtration, such as sepharose
4B etc. may be used. The material of the carrier may be polystyrene, polyacetate, polycarbonate, plastic such as acrylonitrile-butadiene-styrene copolymer, paper such as cellulose, or any other material conventionally used in immunoassays. Further, binding of the antibody to the carrier can be achieved by a known technique such as physicochemical adsorption or chemical binding.

As the labeled antibody, the antibody is a radioactive isotope such as ³ H, ¹⁴ C, ¹²⁵ I, ¹³¹ I, peroxidase, alkaline phosphatase,
An enzyme such as β-galactosidase, a fluorescent substance of fluorescein, a rhodamine B, a luminescent substance such as an isoluminol derivative, and the like can be obtained by labeling with a labeling agent generally used in the art according to a conventional method.

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

First, a certain amount of a sample to be measured is placed in a reaction container, and a certain amount of a buffer solution containing 0.1 to 5%, preferably 0.5 to 2% BSA of maggot serum albumin (BSA), bovine γ-globulin or animal serum is added and mixed. To do. The buffer solution in this case is 0.01
~ 0.5M borate buffer (pH 7.2 ~ 9.0), 0.01 ~ 0.5M phosphate buffer (pH 5.2 ~ 8.5), 0.01 ~ 0.5M veronal buffer (pH 6.8 ~ 9.6) Or a buffer such as 0.01-0.5M Tris-HCl buffer (pH 7.2-9.1), or 0.05-0.
The above buffer containing salts such as 5M sodium chloride and 0.05 to 0.5M potassium chloride is used. Next, an insolubilized antibody (a carrier to which an anti-γ-Sm antibody or an anti-α ₁ -antitrypsin antibody is bound) is added to a mixed solution of the sample and the buffer, and the mixture is reacted for a certain period of time. After the reaction, the carrier is optionally washed, and then the labeled antibody dissolved in the above-mentioned buffer solution (when the insolubilized antibody is an anti-γ-Sm antibody, the anti-α ₁ -antitrypsin antibody and the insolubilized antibody labeled with a labeling agent are In the case of anti-α ₁ -antitrypsin antibody, an anti-γ-Sm antibody labeled with a labeling agent is used) and the carrier is allowed to react for a certain period of time. After the reaction, after washing the carrier,
The amount of the γ-Sm-α ₁ AT complex can be known by detecting the amount of the labeling agent of the labeled antibody bound to the carrier.

Next, according to a test example, γ-Sm- in serum of a prostate cancer patient
It will be explained that prostate cancer can be diagnosed by detecting the α ₁ AT complex and measuring only the complex. The γ-Sm monoclonal antibody used in the test examples and the examples described later is described in the literature, clinical tests, 28, pp. 1755-1758, 198.
We used the one made according to 4 years (Shinichi Kamachi et al.).

Test Example 1 Detection of γ-Sm-α ₁ AT complex in serum of prostate cancer patient After adding 30 ml of serum of prostate cancer patient to Sephadex G-100 column (5 × 90 cm), 0.1 M containing 0.9% sodium chloride
Elution was performed with a borate buffer solution (pH 8.0), and 10 ml each was collected. Using 100 μl of each fraction, anti-γ-Sm monoclonal antibody-bound ABS in accordance with the measurement procedure described in the above-mentioned literature, clinical examination, 28, pp. 1755-1758, 1984 (Shinichi Kamachi et al.).
Free γ with beads and peroxidase-labeled anti-γ-Sm antibody
Both -Sm and γ-Sm-α ₁ AT complex were measured together,
On the other hand, only the γ-Sm-α ₁ AT complex was measured using anti-γ-Sm monoclonal antibody-bound ABS beads and peroxidase-labeled anti-α ₁ -antitrypsin antibody according to Example (4) described later. The results are shown in Fig. 1. From these results, according to the above-mentioned literature, clinical examination, 28, 1755-1758, 1984 (Shinichi Kamachi, et al.), When anti-γ-Sm antibody was used for both insolubilized antibody and enzyme-labeled antibody, free γ −Sm and γ−
Both of the Sm-α ₁ AT complexes were detected, while different antibodies (anti-γ-Sm antibody and α ₁
-Antitrypsin antibody)
Only the α ₁ AT complex can be detected.

Test Example 2 Relationship between γ-Sm-α ₁ AT complex in serum and prostate cancer γ-sera in serum of 19 normal subjects, 19 patients with benign prostatic hyperplasia and 19 patients with untreated prostate cancer The Sm-α ₁ AT complex 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-α ₁ AT complex in the serum of prostate cancer patients was higher than that in normal subjects and patients with benign prostatic hyperplasia. As is clear from the above test examples, the measurement of the γ-Sm-α ₁ AT complex of the present invention is useful for the diagnosis of prostate cancer.

Next, the present invention will be described with reference to examples.

Example (1) Preparation of insolubilized antibody Anti-γ-Sm monoclonal antibody or anti-α ₁ -antitrypsin antibody was adjusted to 15 μg / ml with 0.1 M borate buffer (pH 8.0) containing 6% sodium chloride. Spherical (diameter 6.35 mm)
Wash the acrylonitrile-butadiene-styrene (ABS) beads with a detergent and distilled water thoroughly to
100 ml of ABS beads were immersed in ml. After leaving it at 37 ℃ for 24 hours,
The ABS beads were washed with 9% sodium chloride solution, immersed in 0.1 M borate buffer (pH 7.4) containing 1% bovine serum albumin, 0.1% sodium azide and 0.9% sodium chloride, and stored at 4 ° C.

(2) Preparation of enzyme-labeled antibody i) Peroxidase-labeled anti-γ-Sm antibody F (ab ′) ₂ was purified from anti-γ-Sm rabbit serum by a conventional method, and reduced with 2-mercaptoethylamine / hydrochloric acid to produce Fa.
got b '. Next, a maleimide group was introduced into peroxidase using N-hydroxysuccinimide ester of N- (4-carboxycyclohexylmethyl) maleimide, and the thiol group of Fab 'was reacted to prepare a peroxidase-labeled anti-γ-Sm antibody. .

ii) Peroxidase-labeled anti-α ₁ -antitrypsin antibody A peroxidase-labeled anti-α ₁ -antitrypsin antibody (Goat, manufactured by Kappel) was diluted and prepared.

(3) Preparation of γ-Sm-α ₁ AT complex 2 ml of γ-Sm solution (2.6 mg / ml) was added to 6 ml of human serum, and the mixture was incubated at 37 ° C.
After standing for 48 hours at 60 ° C, the mixture is added to a Sephadex G-100 column (5 x 90 cm) and contains 0.9% sodium chloride.
Elution was performed with 0.1 M borate buffer (pH 8.0), and 5 ml each was collected. Fractions containing γ-Sm-α ₁ AT complex were collected and
The monoclonal antibody was applied to a Sepharose 4B column (1 × 10 cm). The column was washed with about 50 ml of 0.1 M borate buffer (pH 8.0) containing 0.5% sodium chloride, and then eluted with 0.1 M borate buffer (pH 8.0) containing 3.5 M potassium thiocyanate, 3 ml aliquots were collected. γ-Sm-α ₁ A
Fractions containing T-complex were collected, concentrated (about 5 ml), and 0.9 M sodium chloride in 0.1 M borate buffer (pH 8.0) 5
It was dialyzed against. After dialysis, it was added to a Sephadex G-100 column (2 × 90 cm) and eluted with 0.1 M borate buffer solution (pH 8.0) containing 0.9% sodium chloride, and 3 ml aliquots were collected. Fractions containing γ-Sm-α ₁ AT complex were collected and concentrated to obtain γ-Sm-α ₁ AT complex.

The absorbance at 280 nm was measured, and the concentration showing the absorbance of 1.000 was defined as 1 unit / ml of γ-Sm-α ₁ AT complex.
Based on this, a standard solution of the γ-Sm-α ₁ AT complex in Example (4) was prepared.

(4) Measurement of γ-Sm-α ₁ AT complex 200 μl of 0.1 M borate buffer (pH 7.4) containing 1% bovine serum albumin and 0.3 M sodium chloride was placed in a test tube of 100 μl of sample or standard solution. Was added and stirred. Anti-γ-
Insert one Sm monoclonal antibody-bound ABS bead at 4 ℃
I left it for 15 hours. Saline containing 0.05% Tween 20
The ABS beads were washed twice with 4 ml. Peroxidase-labeled anti-α ₁ -antitrypsin antibody (1% bovine serum albumin, 0.1 M borate buffer containing 0.3 M sodium chloride, pH
(Prepared by diluting with 7.4) 300 μl was added and left at room temperature for 2 hours. The ABS beads were washed with 4 ml of saline containing 0.05% Tween 20. Take 0.5 ml of citric acid-phosphate buffer (pH 6.0) containing 0.2% O-phenylenediamine dihydrochloride and 0.015% hydrogen peroxide in a new test tube, put the washed ABS beads in it, and let it stand at room temperature. Left for 1 hour. 2N sulfuric acid 2.0
Add ml and stir, measure the absorbance at 492 nm,
The concentration of γ-Sm-α ₁ AT complex in the sample was determined from the standard curve.

[Brief description of drawings]

FIG. 1 is a chromatogram in which free γ-Sm and γ-Sm-α ₁ AT complex in serum of a prostate cancer patient in Test Example 1 were separated. The solid line shows the results of detection using anti-γ-Sm monoclonal antibody as the insolubilized antibody and anti-α ₁ -antitrypsin antibody as the enzyme-labeled antibody. The broken line shows the results of detection using anti-γ-Sm antibody for both the insolubilized antibody and the enzyme-labeled antibody. FIG. 2 is a graph showing the γ-Sm-α ₁ AT complex concentration (μUnit / ml) in serum of normal subjects, patients with benign prostatic hyperplasia (BPH) and patients with prostate cancer (PCa).

Claims (4)

[Claims] 1. A method of quantifying a complex consisting of γ-Sm and α ₁ -antitrypsin present in a test sample by a sandwich immunoassay method, wherein an anti-γ-Sm antibody and an anti-α ₁ -antitrypsin antibody are used. A method for quantifying the complex, which comprises: 2. The method according to claim 1, wherein one of the anti-γ-Sm antibody and the anti-α ₁ -antitrypsin antibody is insolubilized and the other is a labeled antibody. . 3. A γ-comprising at least two of an anti-γ-Sm antibody and an anti-α ₁ -antitrypsin antibody.
A reagent for quantifying a complex consisting of Sm and α ₁ -antitrypsin. 4. The quantification according to claim 3, wherein _{one of the} anti-γ-Sm antibody and the anti-α ₁ -antitrypsin antibody is insolubilized and the other is a labeled antibody. Reagent.