JP2617712B2 - Assay method for human prostate specific antigen - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel method for quantifying human prostate-specific antigen by a sandwich immunoassay.

[Background of the Invention]

Human prostate-specific antigen (hereinafter abbreviated as PA) is T.
A protein found in human prostate tissue or seminal plasma by M. Cheu et al. (MCWang et al; Invest Urology, 17 , 159, 1).
980; Oncology, 39 , 1, 1982), a prostate acid phosphatase reported by Cheu et al. (TMCheu et al; Invest
Urology, 15 , 319, 1978). PA is not found in other organs and tissues, and is said to be present only in the human prostate. Therefore, prostate disease,
In particular, it has been particularly noted in recent years because it has a high possibility of being used as a marker for prostate cancer.

At present, the method of measuring prostatic acid phosphatase is mainly used for diagnosis of prostate cancer and observation of the course of treatment, but it cannot be said that the clinical condition sufficiently reflects the disease state. It is desired to establish a reliable diagnostic index and a measurement method thereof.

Reports of diagnosis and observation of the course of treatment using PA as a diagnostic index include, for example, Kuriyama et al; Cancer Research, 4
0, 4658,1980, Kohyo Sho 57-500169 JP, Pontes et al;
J. Urology, 128 , 1216, 1982; Ogawa et al .; Radioisotopes, 33 , 2
73, 1984, etc., and it has been reported that measuring the fluctuation of blood PA is effective for diagnosing prostate cancer and observing the course of treatment. However, the measurement methods used in these reports are at once short in terms of both sensitivity and specificity, and have problems in reproducibility, and are very time-consuming (usually more than 24 hours). ) And its operation is very complicated, but various problems remain in its practical application even though its effectiveness can be understood.

[Object of the invention]

The present invention has been made in view of the above-mentioned circumstances, and has a high sensitivity, excellent specificity, excellent reproducibility, simple operation and extremely short measurement time, novel quantification of PA by a sandwich-type immunological assay. Is to provide a law.

[Configuration of the invention]

In order to achieve the object of the present invention, the present invention has the following configuration.

"In the quantification of PA by sandwich immunoassay, PA produced by hybridomas formed by fusion with cells from the tumor line of mice and splenocytes from mice previously immunized with PA, Monoclonal Anti-PA with Specificity for Antibodies and Immunoglobulin Class of IgA
A combination of an antibody and a polyclonal anti-PA antibody obtained from an animal immunized with PA.
Quantitation method. That is, the present inventors have proposed the method of Cheu et al. (Oncology, 39 , 1, 1).
1982) Monoclones with high specificity for PA produced by hybridomas formed by fusion of splenocytes from mice previously immunized with PA purified from human seminal plasma and cells from a mouse tumor line As a result of intensive research, to assemble a PA measurement method using a sandwich-type immunoassay method that is highly sensitive, specific, and reproducible, and that is extremely practical and easy to use, using an anti-PA antibody, PA specific and immunoglobulin class IgA
The present inventors have found that the object of the present invention can be achieved by using a monoclonal anti-PA antibody that is a polyclonal anti-PA antibody obtained from an animal immunized with PA in combination with the monoclonal antibody, and have completed the present invention. .

The monoclonal anti-PA antibody used in the present invention (hereinafter referred to as PA
-Abbreviated as MCA. ) Is a conventional method, that is, G. Khler and C. Milstein; Nature, 256 , 49
According to the cell fusion method established according to US Pat. No. 5,1975), a hybridoma is produced by fusing cells from a tumor line of a mouse with splenocytes of a mouse previously immunized with PA, and a monoclonal clone produced from the hybridoma is produced. A monoclonal anti-PA antibody which is selected from those obtained by collecting an anti-PA antibody by a conventional method and which is particularly suitable for the purpose of the present invention, that is, a monoclonal anti-PA antibody whose immunoglobulin class is IgA is used.

In addition, the polyclonal anti-PA antibody (hereinafter, abbreviated as PA-PCA) used in the present invention can be obtained by a conventional method, for example, “Introduction to Immune Experiments, 2nd Printing, Nao Matsuhashi, Gakkai Shuppan Center, 1981, etc. Horses, cows, sheep,
Any of animals prepared by immunizing animals such as rabbits, goats, rats and mice with PA can be used without exception.

The method for quantifying PA of the present invention uses the PA-
Except for using a combination of MCA and PA-PCA obtained from an animal immunized with PA, for example, sandwich-type enzyme immunoassay (EIA), radioimmunoassay (RIA), and fluorescence immunoassay (FIA) known per se. This may be performed according to the method described in (1) and the like, and the reagent to be used may be appropriately selected and used according to the reagent used in these methods.

Further, the sandwich immunoassay of the present invention may be, for example, a method in which an immobilized antibody is first reacted with a sample containing PA and then a labeled antibody is reacted therewith, or
In a method in which a sample containing PA is reacted with a labeled antibody and then reacted with an immobilized antibody, a sample further containing PA,
A practical method can be assembled by any of the methods of simultaneously reacting an immobilized antibody and a labeled antibody. However, a more preferred embodiment of the present invention includes a so-called one-step method in which a sample containing PA, an immobilized antibody, and a labeled antibody are simultaneously reacted. That is, by performing the reaction in one step,
Workability is improved, and measurement time can be shortened. The method of the present invention makes it possible to assemble such a one-step measuring method.

In the present invention, as a method for immobilizing PA-MCA or PA-PCA on the surface of a water-insoluble carrier, EIA, R
All immobilization methods known per se generally used in IA or FIA can be mentioned without exception. That is, this may be done by physical adsorption on the solid surface, or chemically, amino, carboxyl, hydroxyl, mercapto,
This can also be performed by utilizing a functional group such as an amide group and coupling with a crosslinking agent.

Examples of the water-insoluble carrier used in the present invention include polymer compounds such as polystyrene, polyvinyl chloride, polyethylene, polypropylene, modified cellulose, and polyacrylamide; and inorganic substances such as glass, silica gel, and metal oxides; Any known carrier usually used in FIA can be used. It is also well known that these are usually used in the form of beads, tubes, disk pieces, fine particles (latex particles), microplates and the like.

However, the most preferred embodiment of the present invention is a method using glass beads as a water-insoluble carrier. That is, by using glass beads as the water-insoluble carrier, operations such as phase separation and washing are extremely simplified and the measurement time can be shortened.

Further, in the present invention, as a method of labeling PA-MCA or PA-PCA with a labeling substance, EIA, RIA or
Labeling methods known per se generally used in FIA (eg, Medical Chemistry Laboratory Course, Vol. 8, supervised by Yuichi Yamamura,
Edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, First
Edition, Soft Science Co., Ltd., 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Shoin,
1982 etc.) without exception.

The labeling substance used in the present invention is an enzyme in EIA, a radioisotope in RIA, and a fluorescent substance in FIA.

In the EIA according to the present invention, examples of the enzyme used as the labeling substance include β-galactosidase, alkaline phosphatase, and peroxidase.
It is not limited to these.

In the present invention, any one of a monoclonal antibody and a polyclonal antibody is used as an immobilized antibody, and which is used as a labeled antibody is optional. However, it is possible to perform a measurement excellent in specificity and reproducibility. It is more preferable to use a monoclonal antibody as an immobilized antibody and a polyclonal antibody as a labeled antibody.

The most preferred embodiment of the labeled antibody used in the present invention is an antibody in which a rabbit anti-PA antibody (class; IgG) is converted to Fab 'and then an enzyme is labeled with maleimide. That is, by using the labeled antibody, it is possible to perform a measurement with higher sensitivity and more excellent specificity and reproducibility.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

〔Example〕

Reference Example 1. Monoclonal anti-human prostate specific antigen antibody (PA
-MCA) and preparation of hybridoma producing the same (1) Immunization An emulsion of 0.1 ml of 10 mM Tris buffer (pH 7.2) in which 50 μg of human prostate specific antigen (PA) was dissolved and 0.1 ml of Freund's complete adjuvant were prepared by Balb / c Mouse (male, 4
Weekly). Four weeks later, 50 μg of human PA dissolved in 0.1 ml of 10 mM Tris buffer (pH 7.2) was administered intraperitoneally.

(2) Cell fusion Three days after the final immunization, the spleen of the mouse was removed. Splenocytes ⁸ and NS-1 10 ⁷ cells polyethylene glycol 0.
5 g, 0.5 ml of MEM medium, and 0.15 ml of DMSO were incubated at room temperature for 1 minute for fusion. Cells were RPMIed with 15% FCS
After suspending in I-1640 medium, the suspension was dispensed into five 96-well plates, and culture was started in a CO ₂ incubator. The next day, 2 drops of HAT medium per well were added. Then, every three days, half of the medium was replaced with HAT medium. The culture supernatant two weeks after the fusion was used for detection of antibody production.

(3) Selection of hybridoma In order to select an anti-human PA antibody-producing hybridoma, each of the above 96-well cell culture supernatants was analyzed by ELISA.

First, 0.05 ml of the purified human PA was dispensed at a concentration of 10 μg / ml into a 96-well plate, and allowed to stand at 37 ° C. for 2 hours to immobilize the antigen on the plate. Tween20 (polyoxyethylene sorbitan monolaurate, trade name of Kao Soap Co., Ltd.)
After washing three times with 10 mM phosphate buffer pH 7.4 containing 0.05% (hereinafter abbreviated as washing solution), a 1% BSA (bovine serum albumin) solution to avoid non-specific adsorption of proteins in the culture supernatant Was dispensed in 0.2 ml portions and left at 37 ° C. for 2 hours. 3 with cleaning solution
After washing twice, 0.05 ml of the above cell culture supernatant was dispensed at 37 ° C.
For 2 hours. HAT medium was used as a negative control. Next, after washing three times with a washing solution, 0.05 ml of a peroxidase-labeled anti-mouse immunoglobulin solution (manufactured by DAKO) was dispensed.
It was left at 37 ° C. for 2 hours. After washing three times with a washing solution, a 0.4% orthophenylenediamine solution containing 0.02% of hydrogen peroxide 0.0
After dispensing 5 ml and reacting at room temperature for 30 minutes, 0.05 ml of 6N sulfuric acid was added to stop the reaction, and OD 490 nm was measured. Hybridomas proliferating in the culture supernatant showing OD more than twice that of the negative control were selected as hybridomas producing anti-human PA antibody.

(4) Monocloning Cloning was performed by the limiting dilution method. A 96-well plate was dispensed so that 0.5 hybridomas were contained in each well.
This operation was repeated twice to complete the monocloning. After examining by ELISA, 10 clones were selected and used for the next antibody production.

(5) Preparation of monoclonal antibody 5 × 10 ⁶ of the above 10 hybridomas were administered intraperitoneally to Balb / c mice (male, 8 weeks old) which had been treated with pristane 10 days before, and about 2 weeks later Ascites was collected.

The ascites was subjected to a 40% saturated ammonium sulfate fractionation, and further column chromatography using DEAE cellulose was performed to obtain PA-MCA.
No.106,120,201-1,202-2,204-1,207-2,2
09-2, 213-1, 214-2, and 303-1 were obtained.

Reference Example 2. Identification of immunoglobulin class or subclass of PA-MCA The immunoglobulin class or subclass of the 10 monoclonal antibodies obtained in Reference Example 1 was identified by ELISA as follows.

(Operation method) Purified PA was immobilized on a 96-well plate by the same operation as in Reference Example 1- (3), blocking was performed with BSA, and then the PA-MCA obtained in Reference Example 1 was purified. 2μg / ml
A 0.05 ml solution was dispensed and allowed to react, and an antibody solution (MILES) for the mouse immunoglobulin class or subclass was further added.
The reaction was carried out by dispensing 0.05 ml of a peroxidase-labeled anti-rabbit immunoglobulin solution (manufactured by DAKO).
ml was dispensed and reacted. After washing three times with a washing solution, a 0.4% orthophenylenediamine solution containing 0.02% of hydrogen peroxide 0.0
After dispensing 5 ml and reacting at room temperature for 30 minutes, 0.05 ml of 6N sulfuric acid was added to stop the reaction, and OD 490 nm was measured. The same operation was performed using a washing solution as a negative control.

(Results) The obtained results are shown in Table 1.

Reference Example 3. Examination of specificity of PA-MCA To examine the specificity of PA-MCA obtained in Reference Example 1,
Cross-reactivity with α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and human chorionic gonadotropin (hCG), which are relatively well measured as tumor markers, was examined.

(Operation method) In the same manner as in Reference Example 1- (3), these were immobilized on a 96-well plate using a 2 μg / ml solution of purified PA, AFP, CEA or hCG, and blocking with BSA was performed.
Next, 0.05 ml of a 2 μg / ml solution of PA-MCA obtained in Reference Example 1
Was dispensed, and 0.05 ml of a peroxidase-labeled anti-mouse immunoglobulin solution (manufactured by DAKO) was dispensed and reacted. After washing three times with a washing solution, 0.05 ml of a 0.4% orthophenylenediamine solution containing 0.02% of hydrogen peroxide was dispensed and reacted at room temperature for 30 minutes. The reaction was stopped by adding 0.05 ml of 6N sulfuric acid, and OD 490 nm was measured. The same operation was performed using a washing solution as a negative control.

(Results) The measurement was performed according to the above operation, and it was found that PA-MCA obtained in Reference Example 1 did not react with AFP, CEA or hCG.

Reference Example 4. Examination of specificity by Western Blotting In order to examine the specificity of PA-MCA obtained in Reference Example 1, polyacrylamide gel electrophoresis (PAGE) and Wester
In combination with the enzyme immunostaining method using n Blotting,
PA in the specimen was detected.

(Operation method) A sample containing PA is prepared by a known method (Nature, 227 , 680, 1970)
Using a slab gel with a gel concentration of 5% to 10% according to
PAGE was performed. The gel after electrophoresis was transferred to a nitrocellulose membrane according to the method of Towbin et al. (Proc. Natl. Acad. Sci., 76 , 4350, 1979). The protein band transferred onto the nitrocellulose was reacted with PA-MCA, further reacted with a peroxidase-labeled anti-mouse immunoglobulin solution (manufactured by DAKO) as a secondary antibody, washed, and then subjected to a diaminobenzidine reaction. Thus, the binding site of PA-MCA was detected.

(Results) As a result of the above operation, each of the PA-MCAs obtained in Reference Example 1 reacted with the band of Cheu et al. Having a molecular weight of 33,000.

Experimental Example 1 Selection of Combination of Anti-PA Antibodies Applicable to EIA A combination of anti-PA antibodies applicable to EIA was selected by ELISA.

(Reagent)-Peroxidase-labeled PA-PCA According to a well-known method (enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, second edition, Kikakushoin, 1982), rabbit anti-PA antibody as Fab 'was maleimide. Peroxidase-labeled PA-PCA (hereinafter referred to as P
Abbreviated as OD-labeled PA-PCA. ) Was prepared.

(Operation method) PA-MCA 10 μg / ml solution 0.0
5 ml was dispensed and allowed to stand at 37 ° C. for 1 hour to immobilize the antibody on the plate. After washing three times with a washing solution, 0.2 ml of a 1% BSA solution is dispensed at 37 ° C. to avoid non-specific adsorption.
Let stand for hours. After washing three times with washing solution, PA solution (100ng / m
l) was dispensed in 0.05 ml portions and allowed to stand at 37 ° C for 1 hour to carry out an immune reaction. After washing three times with the washing solution, 0.05 ml of the POD-labeled PA-PCA solution was dispensed, and allowed to stand at 37 ° C. for 1 hour to react. After sufficient washing with a washing solution, the peroxidase activity remaining on the microplate was measured as follows. That is, 0.05 ml of a 0.4% orthophenylenediamine solution containing 0.02% of hydrogen peroxide was dispensed and allowed to react at room temperature for 30 minutes.
The reaction was stopped by adding 05 ml, and OD490nm was measured. (P
A; the value obtained from the solution of 0 ng / ml was Bl, PA; 100 ng / m
a value that has been example with a solution of l was E _S. (Results) The results are shown in Table 2. Here, ×, Δ, 、, and ◎ indicate the following ranges, respectively.

_{×: 1 ≦ E S / Bl} ≦ 2 △: 2 <E S / Bl ≦ 3 ○: 3 <E S / Bl ≦ 5 ◎: 5 <E S / Bl As is clear from these results, of the 10 types of PA-MCA obtained in Reference Example 1, the combination with POD-labeled PA-PCA has relatively good reactivity when No. 106, 201-1, 202-2,
207-2, 209-2, 213-1 and 303-1. Among them, No. 303-1 whose immunoglobulin class is IgA was found to be particularly good.

Experimental Example 2 Selection of PA-MCA Combination Applicable to EIA From the results of Experimental Example 1, PA-MCA7 with relatively good reactivity
Species (Nos. 106, 201-1, 202-2, 207-2, 209-2, 21
Using 3-1 and 303-1), a combination of PA-MCA applicable to EIA was selected.

(Reagent)-Peroxidase-labeled PA-MCA According to a known method (enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd edition, Medical Shoin, 1982), PA-MCA with Fab '(however, No. .303-1) was combined with peroxidase by the maleimide method to prepare peroxidase-labeled PA-MCA (hereinafter abbreviated as POD-labeled PA-MCA).

(Operation method) The same operation method as that of Experimental example 1 was used, except that POD-labeled PA-PCA was replaced with POD-labeled PA-MCA.

(Results) The results are shown in Table 3. (Here, ×, Δ, ○, ◎ are the same as above.) From this result, the combination of PA-MCAs is N
Combinations of Nos. 106 and 303-1 and Nos. 213-1 and 303-1 were good, and in particular, combinations of Nos. 213-1 and 303-1 were considered to be applicable to EIA.

EXPERIMENTAL EXAMPLE 3 A combination of the antibody according to the present invention applicable to EIA obtained from the results of Experimental Examples 1 and 2, and PA using a combination of conventionally used PA-PCA and POD-labeled PA-PCA
A calibration curve was created by the measurement method, and its practicality was examined.

(Reagent)-Preparation of anti-PA antibody-immobilized glass beads Glass beads were immersed in a 2% 3-aminopropyltriethoxysilane-acetone solution at room temperature for 24 hours to carry out aminoalkylsilane conversion. Next, the beads were immersed in a 25% glutaraldehyde solution and activated at room temperature for 4 hours, and then PA-MCA No. 303-1 according to the present invention or PA-PCA obtained from a rabbit.
In a 100 μg / ml solution at 4 ° C. overnight. After washing, the plate was immersed in a 20 mM phosphate buffer containing 5% sucrose and 1% BSA at 4 ° C. overnight, dried, and dried with anti-PA antibody-immobilized glass beads (hereinafter, referred to as “beads”).
Abbreviated as A-PA-G. ).

Preparation of Peroxidase-Labeled Anti-PA Antibody PA-MCA No. 213-1 as Fab 'according to a known method (enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd ed., Medical School, 1982). Or PA-PCA obtained from rabbits
Is bound to peroxidase by a maleimide method to give a peroxidase-labeled anti-PA antibody Fab '(hereinafter, POD-PA-Fa
Abbreviated as b '. ) Was prepared.

(Operation method) 100 μl of a PA solution having a predetermined concentration, 400 μl of POD-PA-Fab ′ and one A-PA-G were placed in a test tube and reacted at 37 ° C. for 2 hours. After washing thoroughly with a washing solution, the activity of peroxidase bound to the beads was measured as follows. That is,
Transfer beads to new test tube and contain 0.02% hydrogen peroxide
Dispense 500 μl of 0.4% orthophenylenediamine solution
After reacting at 37 ° C. for 30 minutes, the reaction was stopped with 2.5 ml of 15N sulfuric acid, and the absorbance at 492 nm was measured immediately.

(Results) The results obtained are shown in FIG. However, here is the solid line Is PA-MCA No.303-1 immobilized A-PA-G and rabbit PA-PCA
In the case of using POD-PA-Fab ′ prepared in combination, the dotted line Are PA-MCA No. 303-1 immobilized A-PA-G and PA-MCA No. 2
In the case where POD-PA-Fab ′ prepared from 13-1 was used in combination, Shows the case where rabbit PA-PCA-immobilized A-PA-G was used in combination with POD-PA-Fab 'prepared from rabbit PA-PCA.

As is clear from this result, the specific PA according to the present invention
-The measurement method using the combination of MCA No. 303-1 and rabbit PA-PCA was found to have the highest sensitivity.

Example 1. Quantification of PA in Blood Blood PA was quantified using the highly sensitive PA quantification method of the present invention assembled based on the results obtained in Experimental Example 3.

(Sample) Serum, normal serum, and a PA solution of a predetermined concentration of each patient of prostate disease, urolithiasis, bladder tumor, urethral tumor, and uterine cancer were used as samples.

(Reagent) PA-MCA No. 303-1 used in Experimental Example 3 was immobilized on A
-PA-G and POD-PA-Fab 'prepared from rabbit PA-PCA were used, and the other reagents were the same as in Experimental Example 3.

(Operation method) Performed in the same manner as in Experimental Example 3, and the serum PA concentration was
It was determined from a calibration curve obtained from the PA solution.

(Results) FIG. 2 shows the relationship between the obtained serum PA concentration and the disease.

As is clear from these results, it was found that the serum PA concentration measured by the PA measuring method according to the present invention was specifically increased in prostate cancer and could be clearly distinguished from other diseases.

In this example, the total measurement time from the reaction to the end of the measurement was 3 to 3.5 hours for each sample.

Example 2. Relationship between serum PA concentration and disease state of prostate cancer Using the PA measurement method according to the present invention, the relationship between serum PA concentration and disease state of prostate cancer was examined.

(Sample) Serum of a prostate cancer patient (relapse, stage A and stage D) and normal serum were used as samples.

(Reagent and operation method) The same procedure as in Example 1 was performed.

(Results) The results are shown in FIG.

As is clear from these results, it was found that the serum PA concentration measured by the PA measurement method according to the present invention well reflects the pathology of prostate cancer.

Incidentally, the total measurement time in each of the samples in this example was 3 to 3.5 hours as in the case of Example 1.

〔The invention's effect〕

As described above, the present invention provides a practical and simple high-sensitivity PA measurement method that reflects prostate cancer and its disease very well, and has high specificity, excellent reproducibility, and the time required for the measurement. Is an invention which has a remarkable effect in that it is within 4 hours, which is a very short time as compared with the conventional one which is 24 hours or more, and it is extremely large clinically for diagnosis of prostate cancer, judgment of therapeutic effect, etc. It is an invention that can be expected to contribute.

[Brief description of the drawings]

FIG. 1 shows a calibration curve obtained in Experimental Example 3, in which the absorbance value (OD) at 492 nm obtained for each PA concentration (ng / ml) on the horizontal axis is plotted along the vertical axis. It is the connection between the points. However, solid line Is a monoclonal anti-human prostate specific antigen antibody (hereinafter, PA-
Abbreviated as MCA. ) No. 303-1 immobilized glass beads and peroxidase-labeled rabbit PA-PCA-Fab 'prepared from rabbit polyclonal anti-human prostate specific antigen antibody (hereinafter abbreviated as rabbit PA-PCA). If used, the dotted line Is PA-MCA No.303-1 immobilized glass beads and PA-MCA N
o. Peroxidase-labeled PA-MCA- prepared from 213-1
In the case where Fab ′ is used in combination, Indicates the case where rabbit PA-PCA-immobilized glass beads and a peroxidase-labeled rabbit PA-PCA-Fab ′ prepared from rabbit PA-PCA were used in combination. FIG. 2 shows the relationship between the blood prostate specific antigen (hereinafter abbreviated as PA) concentration obtained in Example 1 and the disease state. The horizontal axis represents the blood PA concentration in each disease state. This is shown in FIG. FIG. 3 shows the relationship between the blood PA concentration obtained in Example 2 and the disease state of prostate cancer, and the horizontal axis shows the blood PA concentration in each disease state of prostate cancer on the vertical axis. .

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12P 21/08 9162-4B C12N 15/00 C (C12P 21/08 C12R 1:91) (72) Inventor Shinzo Kobatake 1589 Orika Marugabata, Ako-shi 19 Wako Junyaku Kogyo Co., Ltd. Ako Farm (56) References JP-A-59-163565 (JP, A) JP-A-57-500169 (JP, A) )

Claims (4)

(57) [Claims] The present invention relates to a method for quantifying human prostate-specific antigen (hereinafter abbreviated as PA) by a sandwich-type immunoassay, wherein cells from a tumor line of a mouse and mice from a mouse previously immunized with PA are used. A monoclonal anti-PA antibody having specificity for PA and having an immunoglobulin class of IgA, produced by a hybridoma formed by fusion with splenocytes, and a polyclonal anti-PA obtained from an animal immunized with PA A method for quantifying PA, which comprises using an antibody in combination. 2. A combination of a monoclonal anti-PA antibody immobilized on a water-insoluble carrier and a polyclonal anti-PA antibody labeled with a labeling substance such as an enzyme, a radioactive substance, or a fluorescent substance. Item 2. The method according to item 1. 3. A polyclonal anti-PA labeled with a labeling substance.
After making the rabbit anti-PA antibody (class: IgG) Fab ',
Polyclonal antibody labeled with enzyme by maleimide method
3. The method according to claim 2, which is a PA antibody. 4. The method according to claim 2, wherein the water-insoluble carrier is glass beads.