JPH0315700B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a composition for diagnosing prostate disease, and more particularly to a composition for diagnosing prostate disease based on an immunological reaction containing at least an anti-β-MSP antibody. Prostate diseases are very common among the elderly, and among them, benign prostatic hyperplasia and prostate cancer have been on the rise recently. Diagnosis of these prostate diseases is performed by rectal palpation, urethrography, ultrasound diagnosis, pyopsy, etc., but all of these methods impose a painful burden on the patient. Recently, in addition to these methods, prostatic acid phosphatase (hereinafter referred to as "PAP") in serum has attracted attention as a marker for diagnosing prostate diseases. This is based on the report by Gutman et al. [J.Clin.Invest. 17 , 473 (1938)] that PAP increases in serum in prostate diseases, and since then, its application to the diagnosis of prostate cancer and benign prostatic hyperplasia has been It has been done. However, the method of Gutman et al. attempts to determine the abundance of PAP by measuring the activity of PAP. In such cases, it is extremely difficult to determine whether the measured acid phosphatase activity value is due to prostate disease or some other disease, as this increases the This makes it difficult to make an accurate clinical diagnosis. Chu
etc. in the specification of patent application publication No. 55-500034.
Extract and purify PAP and obtain antibodies against it.
By immunochemical method using this,
A method for specifically measuring the amount of PAP is disclosed. However, the current situation is that this method has not achieved the expected detection rate in the diagnosis of prostate diseases. The present inventors obtained human seminal plasma using Se-phadex G-100 gel filtration, Toyopearl HW-55F gel filtration, and column isoelectric focusing, with an isoelectric point of 5.6 and a molecular weight of approx.
10400, a sugar-free protein with approximately 83 amino acids was isolated and purified and named β-Microseminoprotein (hereinafter referred to as "β-MSP"). As a result of examining the interaction of this β-MSP with various body fluids and organ exudates, it was confirmed that β-MSP is one of the prostate-derived seminal plasma-specific antigens since it did not cross with any of them. The present inventors further measured the β-MSP content in the seminal plasma of 10 semen samples using a quantitative method using a multilayer sedimentation reaction, and found that the content was 60 to 90 mg/dl. Next, we used an anti-β-MSP antibody and a second antibody labeled with peroxidase to search for the localization of β-MSP in tissue sections of male reproductive organs using an enzyme-linked immunosorbent method. Specific staining was observed. Similarly, specific staining was also observed in primary prostate cancer cells. In contrast, the bladder mucosa, urethral mucosa, seminal vesicle epithelium, vas deferens epithelium,
No specific staining was observed in tissues such as the testis and bulbar urethra. From these results, β
-MSP was suggested to be a prostate-specific antigen. Based on this, β-MSP can be used as a specific marker in the diagnosis of prostate disease.
We conducted further research because we believed that detecting MSP would enable clinical diagnosis of prostate disease. The present inventors established a radioimmunoassay method for β-MSP and used it to quantify β-MSP in serum in various diseases. On this occasion,
PAP was also measured using the radioimmunoassay method in serum from the same patient. As a result, β
-MSP was not detected in the serum of women.
In addition, the concentration of β-MSP in serum of patients with prostate disease was higher than that of normal human serum.
Particularly in patients with benign prostatic hyperplasia, serum PAP was within the normal range, but β-MSP was increased in many cases. In addition, some patients with prostate cancer had normal PAP but increased β-MSP. The results are shown in FIG. This result suggests that β-MSP is superior to PAP as a specific marker for diagnosing prostate disease. The present invention has been made in view of the above facts. That is, the present invention is a composition for diagnosing prostate disease characterized by containing at least an anti-β-MSP antibody. The anti-β-MSP antibody may be labeled with a labeling substance for radioimmunoassay, oxygen immunoassay, or fluorescent immunoassay, or may be bound to a water-insoluble carrier. By measuring the β-MSP concentration in serum using the composition for diagnosing prostate disease of the present invention, PAP
It can be said that the present invention is extremely useful for diagnosing prostate diseases, since it is possible to detect with high accuracy prostate diseases that were not detected by the method of measuring prostate disease. Specific embodiments and examples of the present invention are shown below, but the present invention applies so-called immune reactions to β-
Any means for detecting MSP is sufficient and is not limited to the following. Specific embodiments of the present invention (1) Composition consisting of β-MSP labeled with a radioisotope (hereinafter referred to as “RI”), anti-β-MSP, an antibody, and a second antibody for B/F separation thing. This is a combination of β-MSP in serum and RI-labeled β.
- It is based on a method that utilizes the fact that MSP competes in binding with anti-β-MSP antibodies.
The RI-labeled β-MSP bound to the anti-β-MSP antibody and the free RI-labeled β-MSP were separated using a second antibody (B/F separation), and then bound to the anti-β-MSP antibody.
By measuring the radioactivity of RI-labeled β-MSP, the amount of β-MSP in serum can be determined, and the presence or absence of prostate disease can be diagnosed from the results. (2) β- labeled with RI, enzyme or fluorescent substance
A composition comprised of MSP and an anti-β-MSP antibody bound to a solid phase. This method is based on the fact that β-MSP in serum and β-MSP labeled with RI, enzymes, or fluorescent substances compete in binding to anti-β-MSP antibodies bound to a solid phase. The amount of β-MSP in serum is determined by measuring the radioactivity, enzyme activity, or fluorescence intensity of the solid phase.
The presence or absence of prostate disease can be diagnosed from the results. (3) A composition consisting of β-MSP bound to a solid phase and an anti-β-MSP antibody labeled with RI, an enzyme, or a fluorescent substance. This method is based on the fact that β-MSP in serum and β-MSP bound to a solid phase compete in binding to a labeled anti-β-MSP antibody. Radioactivity of labeled anti-β-MSP antibody. β- in serum can be determined by measuring enzyme activity or fluorescence intensity.
The amount of MSP is determined, and the presence or absence of prostate disease can be diagnosed from the result. (4) A composition composed of β-MSP sensitized latex and anti-β-MSP antibody. This is a β-MSP sensitized latex and anti-β-
This method is based on the fact that the degree of agglutination of MSP antibodies is inhibited by the presence of β-MSP in serum. −MSP
The amount is determined, and the presence or absence of prostate disease can be diagnosed from the results. (5) A composition comprising an anti-β-MSP antibody sensitized latex. This is based on a method that utilizes the aggregation of β-MSP in serum and latex sensitized with anti-β-MSP antibodies, and can be observed as an agglutination image.
By measuring turbidity, β-
The amount of MSP is determined, and the presence or absence of prostate disease can be diagnosed from the result. (6) Anti-β labeled with RI, enzyme or fluorescent material
−MSP antibody and anti-β− bound to solid phase
Composed of MSP antibody. This method is based on the so-called Sand-Deutsch method, in which β-MSP in serum is bonded to an anti-β
−MSP antibody and then its β-
By further binding labeled anti-β-MSP antibody to MSP, labeled anti-β-MSP bound to the solid phase
The radioactivity, enzyme activity, or fluorescence intensity of antibodies is measured to determine the amount of β-MSP in serum, and the presence or absence of prostate disease is diagnosed based on the results. In this type of diagnostic composition, two anti-β
- MSP antibodies are used, but these antibodies can be used either from the same animal species or from two different animal species (eg goat and rabbit). When using the latter antibodies, one bound to a solid phase and the other labeled, the antibody bound to the solid phase and the label can be combined in a single incubation for the measurement of β-MSP. This is particularly preferred since the immunological reaction between the antibody and β-MSP can be carried out simultaneously. In the specific embodiments of the present invention as described above, representative examples of substances for labeling antigens or antibodies include radioactive isotopes, enzymes, fluorescent substances, latex, and the like. As a radioactive isotope,
¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc. are used, with ¹²⁵ I being particularly preferred. As the enzyme, alkaline phosphatase, malate dehydrogenase, glycose-6-phosphate dehydrogenase, glucose oxidase, glucoamylase, galactosidase, acetylcholinesterase, etc. are used, and peroxidase obtained from horseradish is particularly preferred. As the fluorescent substance, fluorescein isothiocyanate, dansyl chloride, rhodamine B isothiocyanate, etc. are used. Binding of these labels to antigens or antibodies can be achieved by chloramine T if the labeling substance is a radioactive isotope.
method, enzymatic method and Bolton-Hunter method
-Hunter) method, etc., in the case of enzymes, glutaraldehyde, N,N'-0-phenylenemaleimide,
A method using a crosslinking agent such as m-maleimidobenzoyl-N-hydroxysuccinimide ester or a method using a condensing agent, or when the enzyme is peroxidase, the sugar moiety is oxidized to an aldehyde, and the amino group of the antigen or antibody is In the case of a fluorescent substance, a method of adding and mixing the fluorescent substance to the antigen or antibody can be carried out by any method known in this field. Solid phases for binding antigens or antibodies include organic and inorganic polymers (amylase, dextran, natural and modified cellulose, polyacrylamide, agarose, magnetite, porous glass powder,
latex, etc.), the inner walls of test vessels (test tubes, titer plates, cells made of glass or artificial materials, etc.), solid substances (glass or artificial material rods, rods with thick ends, rounded protrusions on the ends) A rod or a ball made into a thin plate is used. Binding of antigens or antibodies to these solid phases can be achieved by physicochemical adsorption methods, by introducing functional groups such as amino groups, carboxyl groups, and halogen groups into the solid phase.
This can be carried out by methods known in the art, such as chemically bonding an amino group or carboxyl group of an antigen or antibody to this. When measuring β-MSP in a specimen using the composition for diagnosing prostate disease of the present invention, the specimen can be used directly or after dilution or pretreatment by an appropriate method. The immunological reaction in the measurement is carried out in an appropriate buffer so as to maintain the optimum pH (4 to 9). Suitable buffers include, for example, phosphate buffer, Tris-HCl buffer, triethanolamine-HCl buffer, and borate buffer. Further, the immunological reaction is preferably carried out at a temperature between 0°C and 55°C. When the label used as an indicator for an immunological reaction is an enzyme, the enzyme activity is measured on a solid phase by a known method, and the substance to be measured (β-
MSP). If the enzyme is a peroxidase from horseradish, the amount of enzyme may be measured based on the catalytic activity present, and this may include hydrogen peroxide and 0-phenylenediamine, 0-dianisidine, as redox indicators. Homovanillic acid, p-cresol, p-
It is measured with aids such as hydroxyphenylethyl alcohol. The invention is illustrated by the following examples. Example 1 Purification of β-MSP The mixed seminal plasma of several people was thoroughly dialyzed against purified water, and the centrifuged supernatant was purified using Sephadex G-100.
(manufactured by Pharmacia) (column 2.6 x 100cm)
0.1M Tris-HCl buffer (PH8.0) containing 0.2M NaCl
Perform gel filtration using a 100% gel filter to collect and concentrate fractions containing β-MSP with a molecular weight of 8,000 to 15,000. Then, using Toyopearl HW-55F (manufactured by Toyo Soda Kogyo Co., Ltd.) (column 1.6 x 100 cm)
Gel filtrate with 0.5% ammonium bicarbonate solution
- Fractions containing MSP with a molecular weight of 8,000 to 15,000 were collected and concentrated, and then subjected to isoelectric focusing (column 110 ml) using Ampholinne (manufactured by LKB) with a pH of 4 to 6 to obtain β-MSP. . This preparation was subjected to 7.5% polyacrylamide gel disk electrophoresis according to a conventional method, and a single band was observed. In addition, as a result of immunoelectrophoresis using antibodies obtained by immunizing rabbits with the preparation using a conventional method and anti-seminal plasma serum obtained by immunizing rabbits with the seminal plasma using a conventional method, it was found that Only one sedimentation line was observed in each case. From this, it was confirmed that the standard sample was β-MSP. Example 2 Anti-β-MSP antibody 1 ml of β-MSP (1 mg/ml) and the same amount of Freund's complete adjuvant were mixed well, and 2 ml of the mixture was dispensed into the footpad of a rabbit. After immunization, approximately 10 days apart The second and third immunizations were administered subcutaneously on the back and thigh, respectively. Blood was collected on the 7th day after immunization, and the serum was examined by Ouchterlony's method, and the presence of anti-β-MSP antibodies was confirmed. Example 3 ¹²⁵ I labeling of β-MSP [ ¹²⁵ I] Bolton-Hunter
Benzene is distilled off from 500 μCi of Hunter's reagent (manufactured by Amersyam, benzene solution) using nitrogen gas. Meanwhile β in 0.1M borate buffer (PH8.5)
-Dissolve MSP to 0.5mg/ml and add 5μ of the solution to the Bolton-Hunter reagent,
React in an ice bath (0°C). To avoid excess reagent binding with the gelatin added later
0.1M borate buffer containing 0.2M glycine (PH
Add 0.5 ml of 8.5) and react (0°C, 5 minutes).
The reaction mixture was transferred to Sephadex G-
25 (manufactured by Pharmacia) (column 1 x 20 cm) is 0.9
Contains % Sodium Chloride and 0.1% Gelatin
Elute using 0.05M phosphate buffer (PH7.4),
Fractionate β-MSP labeled with ¹²⁵ I. Example 4 Quantification of β-MSP in serum Sample serum 0.2ml, diluted anti-β-MSP serum 0.1ml,
0.1 ml of ¹²⁵ I-labeled β-MSP (approximately 20000 cpm/0.1 ml) and 0.09% sodium chloride, 0.1% gelatin,
Mix 0.5 ml of 0.05 M phosphate buffer containing 0.05 M ethylenediaminetetraacetic acid disodium salt, leave at 4°C for 16 to 24 hours, and add 0.2 ml of second antibody (Immuno beads; manufactured by Bio-Rad). ml and leave at 37℃ for about 2 hours. Next, 3.0 ml of 0.9% sodium chloride aqueous solution was added and centrifuged at 1000 g for 10 minutes to perform B/F separation. The radioactivity of the precipitate was measured using a γ-counter. Determine the amount of β-MSP in serum. Example 5 β-MSP concentration in serum β-MSP was measured in the serum of patients with benign prostatic hyperplasia, prostate cancer (before treatment), and diseases other than prostate and in normal human serum according to the method of Example 4. .
The results are shown in the table.

[Table] Example 6 Enzyme-labeled anti-β-MSP antibody Method of Nakane et al. [J.Histochem.Cytcohem.,
22, 1084, (1974)], a peroxidase-labeled anti-β-MSP antibody was produced. That is, peroxidase (manufactured by Sigma,
Type) 10mg, 2ml of 0.3M sodium bicarbonate
After dissolving in the solution, 0.2 ml of 1% fluorodinitrobenzene (ethanol solution) was added, and the mixture was stirred at room temperature for 60 minutes. Then 2ml of 0.08M sodium periodate
was added and stirred for 30 minutes, and then 2 ml of 0.16M ethylene glycol was added and reacted for 60 minutes. Next, this reaction solution was centrifuged at 1000g for 10 minutes, and the supernatant was
Activated peroxidase was obtained by dialysis with 0.01M sodium carbonate buffer (PH9.5) at 4°C for 20 hours. On the other hand, IgG was fractionated from the anti-β-MSP serum using DEAE Affigel Blue (manufactured by Bio-Rad), 10 mg of the IgG was dissolved in 1 ml of sodium carbonate buffer (0.01M, PH9.5), and the same buffer After dialyzing the solution for 20 hours at 4°C, this was mixed with the activated peroxidase solution and allowed to react at room temperature for 3 hours. Furthermore, in order to separate free peroxidase and peroxidase-labeled anti-β-MSP antibody, 0.01 M phosphate buffer (PH7.4) was added as an eluent using Sephadex G-200 (manufactured by Pharmashima Co., Ltd., column 2 x 80 cm). A peroxidase-labeled anti-β-MSP antibody was obtained by gel filtration. Example 7 Antibody-attached polystyrene ball IgG obtained from anti-β-MSP serum was dissolved in 0.01M phosphate buffer (PH7.4) to a concentration of 1 mg/ml. Add ethanol and 20ml of this IgG solution.
Soak 50 polystyrene balls (1/4 inch) washed in 0.01M phosphate buffer (PH7.4) at 4°C.
The mixture was allowed to react for 18 hours. Thereafter, the cells were washed four times with 0.01M phosphate buffer (PH7.4), and then placed in 0.01M phosphate buffer (PH7.4) containing 1% bovine albumin (Sigma) and 0.05% sodium titrate. 4℃
Saved with. Before use, wash with 0.01M phosphate buffer (PH7.4). Example 8 Measurement of β-MSP concentration in serum by enzyme immunoreaction 0.1% bovine albumin (manufactured by Sigma) and 0.05% Triton-X100 in a plastic tube
0.5ml of 0.01M phosphate buffer containing (manufactured by Wako Pure Chemical Industries)
Add 0.1 ml of serum and stir, then add one polystyrene ball coated with antibody and allow to react at 37°C for 1 hour.
Next, after washing the polystyrene ball with the above buffer, 0.5 ml of the above buffer and 0.1 ml of peroxidase-labeled anti-β-MSP antibody are added and reacted at 37°C for 1 hour. Next, after washing the polystyrene ball with the above buffer, place it in another plastic tube.
Contains 0.1% 0-phenylenediamine hydrochloride
Add 0.5 ml of 0.05M phosphate buffer (PH7.2) and 0.5 ml of 0.003% hydrogen peroxide solution, and react at 37°C for 1 hour. Thereafter, 2 ml of 0.1N sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured to determine the β-MSP concentration from the calibration curve.

[Brief explanation of the drawing]

Figure 1 shows serum PAP of 26 patients with prostate disease.
The results of measuring the concentrations of and β-MSP are shown. The figure shows the upper limit of the normal value of PAP. or β−
Indicates the upper limit of the normal value of MSP.

Claims (1)

[Scope of Claims] 1. A protein (β-Microseminoprotein (“β-
Anti-β-
A composition for diagnosing prostate disease, comprising an MSP antibody. 2. The composition according to claim 1, wherein the anti-β-MSP antibody is labeled with a labeling substance for radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay. 3. The composition of claim 1, wherein the anti-β-MSP antibody is bound to a water-insoluble carrier.