JPH0875737A - Method and kit for measuring composite of human prostatic specific antigen and human protein c inhibitor - Google Patents

Abstract

PURPOSE: To obtain an index for discriminating between prostatic hypertrophy and prostatic cancer by accurately measuring a composite of a human prostatic specific antigen(PSA) and a human protein C inhibitor (PCT) in human blood. CONSTITUTION: A PSA/PCI composite in human blood is measured by enzyme immunoassay by a two-antibody sandwich method using a polyclonal antibody or monoclonal antibody to PSA and a monoclonal antibody to PCI. Since this method has high specificity with respect to the prostate and shows a high numerical value only with respect to prostatic hypertrophy and shows a low numerical value with respect to a healthy person and a prostatic cancer patient, the discrimination between prostatic hypertrophy and prostatic cancer heretofor difficult only by the measurement of PSA can be made easy. That is, when PSA concn. shows a high value and the concn. of the PSA/PCI composite shows a high value, prostatic hypertrophy is doubted, and when PSA value shows a high value and the concn. of the PSA/PCT composite shows a low value, prostatic cancer is doubted.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to human prostate specific antigen (Prostate Specific Antigen) in human blood.
n: a complex of human protein C inhibitor (hereinafter abbreviated as PCI) (hereinafter abbreviated as PSA)
(Abbreviated as SA / PCI complex), and more specifically, measuring PSA / PCI complex in human blood that plays an extremely important role in differentiating between benign prostatic hyperplasia and prostate cancer. On how to do.

[0002]

BACKGROUND ART The human prostate undergoes atrophic changes in its 50s, and fibromuscular or glandular nodules appear in the gland (internal gland) around the urethra, gradually forming large nodules. become. Due to the formation of such nodules, the prostate gradually grows and becomes prostatic hypertrophy. Nodule formation increases with aging. For benign prostatic hyperplasia, usually digital rectal examination, residual urine volume measurement, X-ray examination, endoscopy,
Diagnosis is performed by ultrasonic examination, etc., but a biochemical test method specific to benign prostatic hyperplasia has not yet been found, and therefore, a simple test method using blood etc. as a sample is required. There is.

Prostate cancer is the most common type of urinary tract tumor, and although anti-androgen therapy is temporarily successful, it is a disease that is difficult to treat after becoming refractory. Discovery and root cures are needed. Therefore, the introduction of screening for this disease in resident medical examinations and general medical examinations is highly desired. Tumor markers have been used as a means of efficiently diagnosing this disease. Currently used prostate cancer marker is PS
This PSA has high sensitivity for prostate cancer diagnosis and has been put to practical use as the most useful tumor marker for prostate cancer. However, while PSA has a high sensitivity to prostate cancer, it also has a certain sensitivity to benign prostatic hyperplasia, and thus cannot be simply distinguished from benign prostatic hypertrophy. Therefore, there is an urgent need for a clinical examination method capable of distinguishing between benign prostatic hyperplasia and prostate cancer.

The present inventors sought a biochemical test method specific for benign prostatic hyperplasia and capable of differentiating and diagnosing benign prostatic hyperplasia and prostate cancer, and found that the PSA / PCI complex in human blood was accurate. It has been found that a good measurement can provide an index for distinguishing the above-mentioned prostatic hypertrophy and prostate cancer.

[0005]

DISCLOSURE OF THE INVENTION The present invention is a method for measuring PSA / PCI complex in human blood using human blood as a sample,
Using a polyclonal antibody or a monoclonal antibody against PSA and a monoclonal antibody against PCI,
The present invention provides a method for measuring PSA / PCI complex in human blood, which is characterized in that it is measured by an enzyme immunoassay by the two-antibody sandwich method. As described above, conventionally, PSA in human blood has been regarded as one of the most useful indicators of prostate cancer because the measurement shows a high value in prostate cancer. Similarly, in both hypertrophy and prostate cancer, the measurement values are not necessarily specific to prostate cancer because they are localized in the prostate tissue (prostate epithelium) and seminal plasma. Moreover, PSA concentration correlates with the weight of prostate tissue in both benign prostatic hyperplasia and prostate cancer, and shows high values in both of these diseases, but it is difficult to distinguish between benign prostatic hyperplasia and prostate cancer only by the measurement. Therefore, it has been earnestly desired to develop a biochemical test method that can be used as an index for distinguishing both diseases in the technical field.

According to the PSA / PCI complex measuring method of the present invention, the specificity for the prostate is high, the high value is shown only in prostatic hypertrophy, and the low value is shown in the healthy subjects and the patients with prostate cancer. It is possible to easily distinguish between benign prostatic hyperplasia and prostate cancer, which were difficult only by PSA measurement. That is, when the PSA concentration is high and the PSA / PCI complex concentration is high, prostatic hypertrophy is suspected, the PSA concentration is high, and the PSA / PCI complex concentration is low. In some cases, prostate cancer is suspected. As described above, the assay method of the present invention facilitates the differentiation between benign prostatic hyperplasia and prostate cancer, which has been difficult to measure by PSA alone, and is extremely useful as an index in the diagnosis of prostate cancer. .

[0007] PSA / PCI in human blood according to the present invention
The method for measuring the complex is characterized by using a double antibody sandwich method and an enzyme immunoassay method using a polyclonal antibody or a monoclonal antibody against PSA and a monoclonal antibody against PCI. A typical sandwich method of the method of the present invention includes the following methods. That is, when the PSA / PCI complex in human blood is measured by an enzyme immunoassay method using the two-antibody sandwich method, a polyclonal antibody or a monoclonal antibody against PSA is used as a solid-phase antibody, and an enzyme-labeled antibody is used. PS in human blood characterized by using a monoclonal antibody against PCI
This is a method for measuring the A / PCI complex. The present invention will be described in detail below.

The anti-PSA antibody used in the method of the present invention is
Either a polyclonal antibody or a monoclonal antibody may be used, and Wang, M. et al. C. , Et al. : In
vest. Urol. , 17, 159 (1979), PSA purified by an ordinary method can be used as an antigen. Further, a commercially available anti-PSA polyclonal antibody or anti-PSA monoclonal antibody can also be used. Of these antibodies, those capable of binding to the PSA / PCI complex are selected.

The anti-PCI monoclonal antibody used in the method of the present invention is produced, for example, as follows. Barium salt was added to human plasma to remove vitamin K-dependent proteins such as PC, PEG-6000 was added to the supernatant, and the precipitate was collected and eluted, ammonium sulfate fractionation, DEA
E-Sephacel, Dextran Sulfat
e Agarose, Chromatography,
Ultrogel AcA-44, DEAE-Seph
PCI by acel, Chromatography
To purify. The purification method is described in Suzuki, K .; et a
l. J. Biol. Chem. , 258, 163 (1
983).

[0010] The purified PCI thus obtained was intraperitoneally administered to Balb / c mice together with Freund's complete adjuvant, and spleen cells were excised, and myeloma cell line and spleen cells were isolated by Oi and Herzenberg.
(Oi, VT and Herzenberg, L .;
A. , In: Michel, B .; B. and Shi
igi, S. M. , Eds. , Selected Me
ways in Cellular immunol
ogy, San Francisco, CA: WH F
reeman & Co. , 351, 1980) to perform cell fusion. Next, a hybridoma that reacts with PCI can be cloned by the limiting dilution method to establish an anti-PCI antibody-producing cell line to obtain an anti-PCI monoclonal antibody. Of these monoclonal antibodies, those capable of binding to the PSA / PCI complex are selected.

Next, the details of the enzyme immunoassay for PSA / PCI complex using the two-antibody sandwich method in the present invention will be described. The components of the entire measurement system are a solid phase carrier, an anti-PSA antibody for solid phase coating (solid phase), a standard antigen, an anti-PCI monoclonal antibody for labeling (labeling antibody), an enzyme and a substrate. The antibody as it is, or F (ab ′) ₂ obtained by digesting the antibody with pepsin, Fab ′ obtained by reducing F (ab ′) ₂ and Fab obtained by digesting the antibody with papain, etc. The antibody fragment that binds to the antigen can be used as an antibody, or these can be labeled and used as a labeled antibody.

As the solid phase carrier, a solid phase carrier generally used for immunoassay is used. Its shape is a ball,
It may be plate-shaped, rod-shaped, square-shaped, or any other shape, and is a carrier used for gel filtration, such as Sepharose 4B.
And so on. 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. In addition, the binding of the antibody to the solid phase can be achieved by a known technique such as physicochemical adsorption or chemical binding.

Labeled antibodies are ³ H, ¹⁴ C, ¹²⁵
I, ¹³¹ I, and other radioisotopes, peroxidase, alkaline phosphatase, β-galactosidase, and other enzymes, fluorescein, rhodamine B, and other fluorescent substances, isominol derivatives, and other luminescent substances, etc. It can be obtained by labeling with the labeling agent used in a conventional manner. The substrate may be appropriately used depending on the selected enzyme. For example, when alkaline phosphatase is selected as the enzyme, p-nitrophenyl phosphate is selected, and when peroxidase is selected, o-phenylenediamine, or A
BTS (2,2'-azino-bis (3'-ethylbenzothiazoline sulfonic acid)) may be used as a color former and hydrogen peroxide may be used as a substrate.

Next, a method for carrying out the present invention using a solid phase antibody and a labeled antibody will be briefly described. First, a certain amount of the sample to be measured is placed in a reaction container, and bovine serum albumin (BS
A), bovine γ-globulin or animal serum 0.1 to 5
%, Preferably 0.5-2% BSA in buffer solution is added in a fixed amount and mixed. The buffer solution in this case is 0.01 to
0.05M 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, 0.01-0.5 M Tris-HCl buffer, p
H7.2-9.1 buffer solution, or 0.05-0.
The above buffer solution containing salts such as 5M sodium chloride and 0.05 to 0.5M potassium chloride is used. Next, the antibody-bound solid phase is added to the mixed solution of the sample and the buffer, and the mixture is reacted for a certain period of time. After the reaction, the antibody binding is optionally washed, and then the labeled antibody dissolved in the above buffer and the antibody binding are reacted for a certain period of time. After the reaction, the amount of PSA / PCI complex can be known by washing the antibody binding and then detecting the amount of the labeling agent of the labeled antibody bound to the antibody-bound solid phase.

Next, the assay kit of the present invention is used directly for carrying out the assay method of the present invention, and the concrete specifications of the assay reagent kit of the present invention are as follows.
That is, the present measurement kit contains an anti-PSA antibody for solid phase and an enzyme-labeled anti-PCI monoclonal antibody as essential components, and the kit contains a standard antigen and an antigen suitable for the convenience of measurement. A diluent, a reaction solution, a substrate, a substrate solution, a reaction stop solution, etc. are attached. They do not limit the invention. As shown by the experimental examples described below, PSA in human blood by the measuring method of the present invention.
It is possible to measure the PCI complex with high accuracy, the PSA used as the most useful tumor marker in existing prostate cancer, and the PSA according to the method of the present invention.
・ In combination with the measurement of PCI complex, if the PSA concentration is high and the PSA / PCI complex concentration is also high, prostatic hypertrophy is suspected, and PSA is detected at high PSA concentration.
-Prostate cancer is suspected when the PCI complex concentration is low. In this way, benign prostatic hyperplasia and prostate cancer can be differentiated and diagnosed.

The effects of the present invention are summarized as follows.
The present invention provides a method capable of accurately measuring the concentration of PSA / PCI complex in human blood by a simple operation, enables the diagnosis of benign prostatic hyperplasia, and further prevents the existing prostate cancer. Of PSA used as the most useful tumor marker in
It is possible to distinguish between benign prostatic hyperplasia and prostate cancer by carrying out the measurement together with the SA / PCI complex. The present invention will be described more specifically with reference to the examples described below.

[0017]

【Example】

Example 1 (1) Preparation of antibody-bound solid phase Anti-PSA polyclonal antibody (The Binding)
(Manufactured by Site) was diluted to 13 μg / ml with 0.1 M carbonate buffer, pH 9.3, 100 μl was added to each well of a 96-well microplate, left overnight at 4 ° C., and then 0.2 M NaCl, 0 was added. .05M EDTA
And 0.05M tris-containing 0.02% thimerosal
After washing 3 times with hydrochloric acid buffer, pH 7.5, containing 2% bovine serum albumin and 0.5% gelatin, phosphate buffer, p
After injecting 150 μl of H7.5, the mixture was allowed to stand at room temperature for 2 hours and washed three times with the above-mentioned buffer solution to prepare an antibody-bound solid phase.

(2) Preparation of enzyme-labeled antibody 1) Preparation of purified PCI 4 liters of fresh plasma contained 10 mM benzamine hydrochloride, 1 mM
Isobutyl fluorophosphate (DFP), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 50 mg /
L Add dystrypsin inhibitor and add 1M
320 ml of BaCl ₂ was added dropwise.

All the following operations were carried out at 4 ° C. After stirring for 1 hour, centrifugation was performed at 5000 rpm for 30 minutes, the supernatant was collected, and 60 g / l of solid phase PEG-6000 was added. 1
After stirring for an hour, the precipitate was centrifuged at 5000 rpm for 15 minutes to discard the precipitate. Furthermore, 60 g of solid PEG-6000 was added to the supernatant.
/ L, and the mixture was stirred for 1 hour and then centrifuged at 5000 rpm for 30 minutes to collect a precipitate. 0.1M NH ₄ C for precipitation
1, 10 mM benzamine hydrochloride, 1 mM DFP and 1
0.05M Tris-HCl buffer containing mM PMSF, p
500 ml of H7.5 was added and dissolved. DEAE-Seph equilibrated with the same buffer used for lysis
It was applied to an arose CL-68 column, and a flow-through fraction was collected. Ammonium sulfate powder is added to the collected solution to 50%
The mixture was saturated, stirred for 1 hour, centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. Further add ammonium sulfate powder to 70% saturation and stir for 1 hour, then rotate at 8000 rpm for 30
After centrifuging for a minute, the precipitate was collected. 0.1M NaCl for precipitation
1, 1 mM benzamine hydrochloride, 0.1 mM DFP, and
0.05 M Tris-hydrochloric acid buffer containing 0.1 mM PMSF, pH 7.5 was added and dissolved, and the mixture was dialyzed against the same buffer. Dextran Sulfate Agaro
It was applied to a se column and ammonium sulfate powder was added to the PCI fraction to make it 70% saturated. Centrifuge at 10,000 rpm for 15 minutes, collect the precipitate, and collect the minimum amount of solution 0.15M
0.05M Tris-HCl buffer containing NaCl, pH
After dissolution in 7.5, Ultrogel AcA 44
It was applied to a column and the PCI fraction was dialyzed against 0.05M Tris-HCl buffer, pH 9.0. After dialysis, DEAE-S equilibrated with 0.05 M Tris-HCl buffer, pH 9.0
Apply to ephacel (0.5 x 10 cm), 0.05
After thoroughly washing with M Tris-HCl buffer, pH 9.0,
Elution with an eluent having a concentration gradient of 0 to 0.5 M NaCl gave purified PCI. The final recovery rate was 10%.

2) Preparation of anti-PCI monoclonal antibody Purified PCI (50 μg) was intraperitoneally administered to two Balb / c mice (female, 8 weeks old) together with the same volume of Freund's complete adjuvant, and 50 μg of each was administered to each mouse. On the 52nd day, each was intraperitoneally administered to the tail vein. Three days later, the spleen cells were extracted and cell-fused with the myeloma cell line SP-2 / 0-Ag14. Cell fusion is polyethylene glycol 40
Method of Oi and Herzenberg (Oi, VT and Herzenberg,
L. A. , In: Michel, B .; B. and S
hiigi, S .; M. , Eds. , Selected
Methods in Cellular immun
logic, San Francisco, CA: WH
Freeman & Co. , 351, 1980). Next, a hybridoma that reacts with PCI was cloned by a limiting dilution method using a 96-well microplate to establish an anti-PCI antibody producing cell line (15 clones). Each obtained clone was administered to pristane-treated Balb / c mice (1 × 10 ⁷ cells / mouse)
The ascites fluid was collected after 1-2 weeks. Then Ig
For the Gl / κ antibody, each ascites was purified by a 40% saturated ammonium sulfate fractionation and a protein A-cellulofine column chromatography operation. Obtained purified antibody (clone no. 16
0-12B5) was used as an enzyme-labeling antibody of the sandwich EIA system.

3) Preparation of HRP-labeled anti-PCI monoclonal antibody 5 mg of the obtained anti-PCI antibody was added to 0.1 M acetate buffer, p
After dialysis against H4.2, 0.2 mg of porcine stomach / pepsin was added, and the mixture was incubated at 37 ° C. for 24 hours to adjust the pH to 7.0, and then applied to an Ultrogel AcA-44 column, and then 0.1 M phosphate buffer, pH 7. Gel filtration at 0 to remove F (ab ') ₂ in 0.1M phosphate buffer, pH
After dialysis to 6.0, 0.1M mercaptoethylamine (0.1M phosphate buffer, pH 6.0), 5mM ED
50 ml of TA was added and incubated at 37 ° C. for 90 minutes. 0.1 M phosphate buffer containing 5 mM EDTA, p
Fab-SH was obtained by dialysis through a Sephadex G25 column equilibrated with H6.0. On the other hand, horseradish peroxidase (abbreviated as HRP) 2 as an enzyme
mg was dissolved in 0.1 M phosphate buffer, pH 7.0,
N-succinimidyl-4- (N-maleimidomethyl)
-Cyclohexane-1-carboxylate 0.7 mg
(Dissolved in N, N-dimethylformamide) was added and incubated at 30 ° C. for 60 minutes. Sephadex G equilibrated with 0.1 M phosphate buffer, pH 6.0
Dialyze through 25 columns to obtain maleimidated HRP
I got Fab-SH and maleimidated HRP were mixed and incubated overnight at 4 ° C., and Ultrogel AcA equilibrated with 0.1 M phosphate buffer, pH 6.5.
Gel filtration was performed using a -44 column to obtain an HRP-labeled anti-PCI monoclonal antibody.

(3) Preparation of Specimen Serum samples were 0.2 M NaCl, 0.1% bovine serum albumin, 0.05% Tween-20, 1 mM EDTA.
And 0.05M tris-containing 0.02% thimerosal
A test sample was diluted 5 times with a hydrochloric acid buffer solution, pH 7.5.

(4) Measurement of Specimen To the antibody-bonded solid phase in (1) above, 100 μl of the test specimen in (3) above is added per well, and the mixture is incubated at room temperature for 2 hours. 0.2 M NaCl, 0.1% bovine serum albumin, 0.05% Tween-20, 1 mNE
After washing 3 times with DTA and 0.05M tris-hydrochloric acid buffer containing 0.02% thimerosal, pH 7.5, the above (2)
100 μl of enzyme-labeled antibody in 1) is added and incubated at room temperature for 60 minutes. 0.2M NaCl, 0.1%
Bovine serum albumin, 0.05% Tween-20, 1m
M EDTA and 0.02% thimerosal included 0.0
After washing 3 times with 5 M Tris-HCl buffer, pH 7.5,
1.33 mg / ml tetramethylbenzidine and 0.3.
Citrate buffer containing 03% hydrogen peroxide, pH 4.65,
Add 100 μl and leave at room temperature for 30 minutes, then add 1N sulfuric acid 5
After stopping the color reaction by adding 0 μl, the absorbance at a wavelength of 450 nm was measured by a spectrophotometer.

(5) Preparation of standard curve (Sample and method) The standard product of PSA / PCI complex is
It is prepared by either of the following methods (a) and (b). a) Purified PCI 100 obtained in (2) -1)
μl (400 μg / ml) and P obtained by the following method
SA 200 μl (200 μg / ml) and 0.05M
Tris-HCl buffer, pH 7.5, 0.1M NaC
The reaction was carried out at 37 ° C. for 4 hours in a 0.1 U / ml heparin solution. The reaction solution was subjected to gel filtration using an Ultrogel AcA-44 column equilibrated with the above buffer solution. Anti-PSA antibody, which is eluted in the high molecular region,
The fraction that reacted with the anti-PCI antibody was designated as PSA / PCI complex. The PSA / PCI complex concentration is determined by the extinction coefficient (P
The absorbance of the SA / PCI complex 1% solution at a wavelength of 280 nm was calculated from 10.0). For PSA, 15 ml of semen obtained from 5 healthy men was diluted 5-fold with 0.05 M Tris-hydrochloric acid buffer containing 10 mM benzamidine, pH 7.5, and applied to a CM-Sepharose CL-6B column to further pass through the fraction. DEAE-Sepharo
The flow-through fraction was collected on a se CL-6B column.
Ammonium sulfate powder was added to the collected solution to 70% to centrifuge at 10,000 rpm for 15 minutes to precipitate the minimum amount of 1.
After dissolving with 0.05 M Tris-hydrochloric acid buffer containing 0 M NaCl, it was applied to an Ultrogel AcA-54 column,
Purified PSA was obtained.

B) Semen obtained from a healthy person was applied to an agarose column to which the anti-PCI monoclonal antibody obtained in (2) -2) above was bound. The bound fraction was applied to a heparin agarose column, and the PSA / PCI complex fraction was concentrated and then fractionated with Ultrogel AcA-44 to prepare a purified PSA / PCI complex. The PSA / PCI complex obtained was prepared to 90 ng / ml and used as a standard antigen solution. Diluting the above standard antigen solution, (3) preparation of the sample, (4) measurement of the sample,
Measurements were made according to the same procedure as in.

(Results) The results are shown in FIG. The horizontal axis represents the concentration of PSA / PCI complex in the test sample, and the vertical axis represents the absorbance value at a wavelength of 450 nm. The measurement sensitivity (M + 2SD) of this calibration curve is 2.8 ng / ml, and the measurement range is 2.
Linearity was observed at 8-90 ng / ml.

(6) Specimen Measurement (Samples and Methods) 14 healthy subjects, 7 patients with benign prostatic hyperplasia
Prostate cancer patient (stage A) 4 cases, prostate cancer patient (stage B) 2 cases, prostate cancer patient (stage)
C) 5 cases, prostate cancer patient (stage D ₂ ) 7 cases,
The serum was measured according to the same procedure as in (3) Preparation of sample and (4) Sample measurement above.

(Results) The results are shown in FIG. Each P in FIG.
The average concentration of SA / PCI complex is 11.4 ng for healthy subjects.
/ Ml, patients with benign prostatic hyperplasia 46.8 ng / ml, patients with prostate cancer (stage A) 49.4 ng / ml, patients with prostate cancer (stage B) 12.7 ng / ml, patients with prostate cancer (stage C) 18.0 ng / ml, prostate cancer patient (stage D ₂ ) 8.4 ng / ml. When the upper limit of normality (M + 2SD) was calculated from the serum value of the healthy subject, it was 22.6 ng / ml, and when the positive rate was calculated from this value, patients with benign prostatic hyperplasia (6/7, 85.7).
%), Patients with prostate cancer (stage A) (2/4, 5)
0.0%), prostate cancer patients (stage B) (0 /
2, 0%), prostate cancer patients (stage C) (1 /
5, 20%), prostate cancer patients (stage D ₂ ) (0
/ 7, 0%), and the positive rate is 85.
It was as high as 7%. However, for prostate cancer,
Since the positive rate was low even in e, it was confirmed that the assay method of the present invention is useful for the diagnosis of benign prostatic hyperplasia.

(7) Comparison with existing marker The same sample as that measured in the above (6) was used as a commercial product PSA.
Serum PSA concentration was measured with a measurement kit (Young Laboratory). The normal upper limit of this kit is 3.2n
It was g / ml.

(Results) The results are shown in FIG. Each PS in Figure 3
The average concentration of A was 2.53 ng / ml in healthy subjects, 9.26 ng / ml in patients with benign prostatic hyperplasia, and in patients with prostate cancer (st
age A) 6.37 ng / ml, prostate cancer patient (st
age B) 7.90 ng / ml, prostate cancer patient (st
age C) 28.9 ng / ml, prostate cancer patient (st
ageD ₂ ) 1117.6 ng / ml, and as can be seen from FIG. 3, the positive rate of patients with benign prostatic hyperplasia is (7 /
7, 100%). Therefore, PSA / PCI
When the complex concentration was compared with the PSA concentration used as a prostate cancer marker, PS at a high PSA concentration
If the A / PCI complex concentration is high, prostatic hyperplasia is suspected, and if the PSA / PCI complex concentration is low, prostatic cancer is suspected.

[Brief description of drawings]

FIG. 1 is a graph showing an example of a standard curve used in the measuring method of the present invention.

FIG. 2 is a graph showing an example of the result of measurement of a sample by the measuring method of the present invention, showing serum P of healthy subjects and patients with various diseases.
It shows the measured value of the SA / PCI complex concentration.

FIG. 3 is a graph showing an example of measurement results with existing markers, showing the measured values of serum PSA concentrations in healthy subjects and patients with various diseases.

Claims (5)

[Claims] 1. A method for measuring a complex of human prostate-specific antigen and human protein C inhibitor in human blood using human blood as a sample, comprising a polyclonal antibody or a monoclonal antibody against human prostate-specific antigen and human protein C. A method for measuring a complex of a human prostate-specific antigen in human blood and a human protein C inhibitor, which comprises measuring by a double antibody sandwich method with an immunoassay using a monoclonal antibody against the inhibitor. 2. When measuring a complex of human prostate-specific antigen in human blood and a human protein C inhibitor by an enzyme immunoassay using a two-antibody sandwich method, it is used as a solid-phased antibody against human prostate-specific antigen. Human prostate specific antigen in human blood and human protein C characterized by using a polyclonal antibody or a monoclonal antibody and a monoclonal antibody against a human protein C inhibitor for an enzyme-labeled antibody
A method for measuring a complex with an inhibitor. 3. A method for diagnosing benign prostatic hyperplasia.
And the measuring method described in 2. 4. A measuring kit for measuring a complex of a human prostate-specific antigen in human blood and a human protein C inhibitor by an enzyme immunoassay utilizing a two-antibody sandwich method, which is for solid phase. An assay kit comprising an anti-human prostate-specific antigen antibody and an enzyme-labeled anti-human protein C inhibitor monoclonal antibody, and optionally a standard antigen, an antigen dilution solution, a reaction solution, a substrate, a substrate solution and / or a reaction stop solution. 5. A method for diagnosing benign prostatic hyperplasia.
The described measurement kit.