JPS5938657A - Composition for diagnosis of prostate disease - Google Patents

Abstract

PURPOSE:To obtain a composition capable of detecting a prostate disease with high accuracy, by containing at least an anti-beta-MSP antibody to measure the concn. of beta-microseminoprotein (beta-MSP) in serum. CONSTITUTION:beta-MSP is obtained from human serum by a gel filtering method and a column isoelectric point electrophoretic method to immunize a rabit and an anti-beta-MSP antibody is obtained. According to a method by Nakane et al., a peroxidase tagged anti-beta-MSP antibody is prepared. After a buffer solution and serum are stirred, an anti-body adhered polystyrene ball are put in the mixed solution to perform reaction. Subsequently, the peroxidase tagged anti-beta-MSP antibody is added to perform reaction while a buffer solution containing O-phenylenediamine hydrochloride is added and absorbency is measured to calculate the concn. of beta-MSP from a calibration curve.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for diagnosing prostate disease, and more specifically to a composition for diagnosing prostate disease that involves an immunological reaction containing at least an anti-β-M8F 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 through rectal palpation.

This is done using methods such as urethrography, ultrasound diagnosis, and biopsy, but all of these methods impose a painful burden on the patient. Recently, in addition to these methods, prostatic I in serum!
! fF phosphatase (hereinafter referred to as "rPAP") is attracting attention as a marker for diagnosing prostate diseases. This is due to the report by Gutman et al. (J.

01in, Invest, 1yu, 473(193[
1)) Since then, Mae, Tate, nl? ] It has been applied to the diagnosis of 111P hypertrophy of the prostrate. However, the method of Gutman et al. attempts to find out the existence of the disease by measuring the activity of FAI', which is a prerequisite for diseases other than prostate disease.
In such cases, it is necessary to determine whether the measured acid phosphatase activity value is caused by a prostate disease or another disease, since serum acid phosphatase that is not immediately derived is found in the blood. This makes accurate clinical diagnosis of prostate disease difficult. Oh +, +, etc. are PAP in the patent application publication specification No. 55-500034.
PAphadex) G in serum was extracted and purified, an antibody against it was obtained, and an immunochemical method using it
100 gel filtration, Toyo-pear
l) By HW-55F gel filtration and Karamoto focused electrophoresis, the isoelectric point was 5.6 and the molecular weight was approximately 10°400.
β-Microsemin$oprote is prepared by preparing a molecule of a sugar-free protein with approximately 83 amino acids.
in (hereinafter referred to as "β-MsP").

As a result of cross-examination of this β-M8P with various body fluids and exudates from the genitals, it was found that β-M8P is one of the prostate-derived seminal plasma-specific antigens. It was confirmed that The inventors further found that 10
Regarding the semen in the example, the β-M S P content J, ? T to f! It was 60 to 90 FW/d after one constant.

Next, using an anti-β-MSP antibody and a second antibody labeled with peroxidase, we searched for the localization of β-Δisp in a tissue piece of the male reproductive organ using an enzyme-linked immunosorbent method.
Specific staining was observed in prostatic epithelial cells and prostatic secretions. Similarly, specific staining was also observed in primary prostate cancer cells. On the other hand, no specific staining was observed in tissues such as the bladder mucosa, urethral mucosa, seminiferous N epithelium, vas deferens epithelium, Sumikyu, and bulbourethral gland. These results suggested that β-MSP is a prostate-specific antigen. From this, we believe that clinical Hh lr# of prostate cancer disease can be determined by using β-MSP as a specific marker for diagnosis of prostate disease and detecting β-8P in serum, and further research is needed. It slumped.

The present inventors established a radioimmunoassay method for β-MSP and used it to detect β-MSP in serum in various diseases.
- 8P was quantified. This 117! PAP was also measured using the radioimmunoassay method using serum from the same patient. As a result, β-MSP was not detected in the female serum.

Furthermore, the r'' value of β-MSP in serum in patients with prostate disease was higher than that in normal human serum.

In the case of benign prostatic hyperplasia, serum PAP was normal, but β-M8F was increased. The results are shown in Figure 1 of Foil. These results demonstrate that β-MSP can be used as a specific marker for diagnosing prostate diseases.
PJ: It shows that it is also excellent l15fT'
It is something to do.

The present invention is based on the above facts.

That is, the present invention provides a composition for diagnosing prostatic Hyp disease, which is characterized in that it contains an anti-β-MSP antibody. PA may be labeled with a marker substance for immunoassay, r1 gamma immunoassay, or t-immunoassay, or may be bound to a water-insoluble carrier.

β- in serum using the composition for diagnosing prostate disease of the present invention
By measuring the M 8 P concentration, it is possible to detect with high accuracy prostate diseases that are not detected by the method of measuring PAP, and therefore, it can be said that the present invention is extremely useful for diagnosing prostate diseases.

Any means for detecting labeled β-M SP, anti-β-M SP antibodies, and B/
Set Q' which is 4%7 h'c from the second antibody for F separation
l'ffnThis is an alternative to the method that utilizes the fact that β-MSP in serum and β-MSP labeled with phosphor I compete with each other in the binding of anti-β-MSP antibodies. anti-β-
The 8P-8P bound to the MSP antibody and the free 8-labeled β-MSr were separated using a second antibody or the like.
'F separation) and R, I combined with anti-β-M S P antibody.
By measuring the radiation of labeled β-MSP, β-It, (8Pf&) in serum can be determined, and the presence or absence of prostate disease can be diagnosed from the results.

(2) Group J7 consisting of RI, r1f or fluorescently labeled β-t, 48P, and anti-β-MSP antibody bound to a solid phase.

This is because β-M8P in serum and R1, β-MSP separated by enzyme or fluorescent substance, combine #:' in the same phase in binding to anti-β-[l]P antibody. This method is based on competition, and the amount of β-M S P in serum is determined by measuring in-phase radioactivity, enzyme activity, or fluorescence 51i, and the presence or absence of prostate disease can be determined from the results. A diagnosis can be made.

(3) β-MSP and RI bound in the same phase, enzyme matabantu, and anti-7! labeled with fluorescent substance. / -M8P
Group I nine products made from antibodies +i, ¥.

This is a combination of β-MSP in serum and β-M bound to a solid phase.
This is based on a method that utilizes competition in binding to the anti-β-M 8 P antibody, which is targeted by SP, and is based on the β-? J Labeled anti-β-MS bound to R3F
P antibody's radioactivity, enzymatic activity, or fluorescence intensity i! t'
β-M S P in serum, J=i
is determined, and the presence or absence of prostate disease can be determined from the results.
:ji can do it.

(4)/-A composition comprising MSr-sensitized latex and anti-β-MSP antibody.

This method is based on the fact that the degree of aggregation between β-M81E' sensitized latex and anti-β-NSP antibody is inhibited by the presence of β-MSI' in serum, and the agglutination image is )? Also, by measuring the degree of u stay,
The presence of β-MSP in serum is determined, and from the results it is possible to diagnose prostate disease.

(5) Silk J containing anti-β-M SP antibody-sensitized latex
y”i.

thing.

This is based on the fact that β-MSP and anti-β-) in the serum and fSP antibody-sensitized latex are lf% (f&).
I-R4,B in IIJ
P nails are determined, and the presence or absence of prostatic 1liil disease can be diagnosed from the results.

(6) Anti-β-Δ labeled with RI, enzyme or fluorescent material
(Composed of SP antibody and anti-β-P48F antibody bound in the same phase.

This is based on the so-called Sandermansch method, which uses anti-β-MSP that binds 7;'-Mar' in serum in the same phase.
The antibody is bound to β-M811, and then f'N is added to the β-M811.
By binding the IIM anti-β-MSP antibody, the radioactivity, enzymatic activity, or fluorescence intensity of the labeled anti-β-ΔIsP antibody bound to immobilized 417 was determined to 1 (IIJ), and the
-h, i 8 P f-'t and from the result,
I+￥! It is used to diagnose the presence of a disease.

In this f.H diagnostic composition, two anti-β-M
8 P antibodies are used, but these antibodies are from the same animal species.
;sui′,) or two different animals fi
(e.g. goat and rabbit) can be used. The latter antibodies, one of which is bound in phase and the other labeled, can be used to
This is particularly preferred in the measurement of MSP because the immunological reaction of the antibody bound to the solid phase and the labeled antibody with β-M 8 F can be carried out simultaneously during a single incubation.

In the specific R1 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. Radioactive isotopes include malic acid dehydratase, glucose-6-phosphorus [・2 dehydration 2F? ! , glucose oxidase, glucoamylase, galactosidase, acetylcholinesterase, etc. are used, and from horseradish to bamboo? ') are preferred. Flumerescein isothiocyanate-1 as a fluorescent substance Fl.
, Dunk Al chloride, rhodamine 3 isothiocyanate, etc. are used.

Binding of these labels to antigens or antibodies (if the label is a radioactive isotope, the chloramine T method, the enzymatic method and the Bolton-Hunter method)
er) method, etc., in the case of enzymes, use glutaric fulldehyde.

A method using a cross-linking agent such as N,N'-0-phenylenemaleimide, 5-maleimidobenzoyl-N-hydovxysuccinimide ester, or a method using a condensing agent, and when the enzyme is peroxidase, to 2
A method of converting the i moiety into an aldehyde to form an aldehyde and bonding it to the amino group of the antigen or antibody, and in the case of a fluorescent substance, a method of adding and mixing the fluorescent substance to the antigen or antibody, etc. are all known in this field. This can be done by the following method.

The solid phase to which the antigen or anti-fedrin is bound can be made of free and silent polymers (amylase, dextran, etc.).

Natural] 3 and 11-mylulose, polyacrylamide.

Agarose r? + Cotton iron scissors, porous glass powder, latex, etc.), trial! -9 Binding of antigens or antibodies in the same phase to the inner wall of a container (test tube, titer plate, cell of glass or artificial material, etc.) is achieved by physicochemical adsorption, 7-min groups, carboxyl groups, etc. on the solid phase. This can be carried out by methods known in the art, such as a method of introducing a functional group such as a halogen group and chemically bonding an amino group or carboxyl group of an antigen or antibody to the functional group.

When measuring β-M8F in columnar 4N using the composition for diagnosing prostate disease of the present invention, the test sample can be used directly, or it may be diluted or pretreated by an appropriate method before use. . Immunological reactions in the Nl assay are carried out in an appropriate buffer to maintain the optimum pH (4-9). Suitable buffers include, for example, phosphorus et al. 9 salt buffer, Tris-hydrochloric acid, etc. tt liquid, Trietano-Rumilly Salt Shunrki liquid, Hon 1 11 mark j's Nsu old and 1fi liquid are extinguished. Further, the immunological reaction is preferably carried out at a temperature between 0°C and 55'C. When the label as an indicator of an immunological reaction is an enzyme, the enzyme activity is measured by a known method on a solid phase and serves as a measure for the amount of the substance (β-R48P) to be measured. 6.2 If the enzyme is a peroxidase from Western Warabi, the enzyme's capacity is better measured based on the catalytic activity present, and this is based on hydrogen peroxide and 0-phenylene as redox indicator. diamine,
0-Dianisidine, homovanillin #1 * Determined with the aid of p-cresol, p-hydroxyphenylethyl alcohol, etc.

The invention is illustrated by the following examples.

Example 1 1'r7t,'! of β-MS P The mixed spermatozoa named z′ was sufficiently dialyzed against purified water, and the centrifuged supernatant was purified using Sephadex G-1.
00 (Pharmacia r!ri (column 2.6 x 10
0 an ) at 0.2 M Nn (0.1 including HA
M) Gel filtration was performed using Lis-HCl buffer (pH 8,0) to obtain molecules j; l B, 0 containing β-M8P.
Fractions from 00 to 15.000 were collected by i kri. Next, Toyopearl HW-
55FC Toyo 9'j Datsuko Co., Ltd.) (Column 1.6
After gel filtration with 0.5% ammonium bicarbonate 1t'i solution at lin
e) Perform isoelectric focusing (column 110WI/+) using (L, B company ij+!) and β-M S
I got P.

This specimen was subjected to 7.5% polyacrylamide gel disk electrophoresis according to a conventional method, and a single band was observed. In addition, anti-sperm I was obtained by immunizing rabbits using the same antibody and seminal plasma obtained by immunizing rabbits using the conventional method. As a result of performing immunoelectrophoresis, we were able to detect only one sedimentation line within each β region.

From this, it is confirmed that the sample is β-MSP.
L, ta.

Example 2 Anti-β-MSP antibody β-MSP (1m!?/Fl (same as 1rpe)
t (1) 7 o India's complete adjuvant is mixed well and its 2m/! Rabbit foot pad (Foot
After dispensing the pipette (pid), the second
The third immunization was performed subcutaneously on the back and thigh, respectively. Blood was collected on the 7th day after immunization, and the results were analyzed using the ophthalmology method, and as a result, the presence of anti-β-meSF antibodies was confirmed.

Real & Ij Example 3゜β-MSI' 125■ Insidious (125I) Bolton -
Benzene was distilled off using nitrogen gas from 500 μC of Hunter's reagent (Amarjam Reiken, benzene solution).

On the other hand, 0.1M salt solution (β-M
isP is dissolved at a concentration of 0.5 μ/me, and 5 μβ of the solution is added to the Porton-7 center sample mulberry and reacted in a water bath (0° C.). Excess trial hollyhock added later, 7
e Add and react (0°C, 5 minutes). The reaction mixture was treated with Sephadex (8epbadex) G-25 (7
(manufactured by Furmacia) (column I x 20 cm)
．． 0 containing 9% sodium chloride and 0.1% gelatin
．． Elute with 0.05M phosphorus saline preoperative solution (117.4), and fractionate β-MSP labeled with 1''5■.

Example 4 Quantification of β-MSP in serum Sample serum 0.2 ml, diluted anti-β-MSp J11
x Sei o, i y Ie1125I labeled β-M8F (approximately 2
0.000cpm/0.1tire) 0.1
mg and 0.09% sodium chloride, 0. IN gelatin, 0.05M ethylenediamine 4G'h m I! 0 and 05 M phosphorus l' each containing sodium salt
：＞＠ Mix 0.5 me of slowing liquid and heat at 4℃ for 16~2
After 4 hours of Pc m , 0.2 Fe of m=antibody (immunobeads; manufactured by Vime-Rad) was added and incubated at 37° C. for about 2 hours. Next, add 4 ml of 0.9% sodium chloride solution3. After adding Or, ・r and centrifuging at 1000F for 10 minutes to separate B/F, the radioactivity of the precipitate was measured with an r-counter, and the sample was determined from the standard line prepared using a standard solution of β-M 8 F. Determining the amount of β-MSP in serum 1.
Example 5 β-M SP in serum 9° β-M SP in normal human serum was measured. The results are shown in the table.

Example 6 Enzyme-labeled anti-β-P11sp antibody Method of Nakane et al. (J, Histochem,
Oytochem, .

22.1084, (1974)), a peroxidase-labeled anti-β-M SP antibody was produced.

No, peroxidase (manufactured by Sigma).

Typed) 10 mg was MiPfied in 0.3 M sodium hydrogen carbonate 2re, 0.2 me of 1χ fluorodinitrobenzene (ethanol solution) was added, and the mixture was stirred at room temperature for 60 minutes. Then, 0.08 N4 sodium periodate, 2 me was added for 30 minutes (stirred for 1 J, and 0.1 nM ethylene glycol was added for 2 r.
/! and reacted for 6 minutes. Next, this reaction solution was spun at 1.000 f for 1 o, and the supernatant was incubated at o, o.
i p, charcoal-based sodium hydroxide solution (pl+ 9.5)
Activated vermexidase was obtained by dialysis at 4° C. for 20 hours.

On the other hand, IgG was fractionated using anti-β-MSP serum I) EAE Affigel Flu (manufactured by Bioken Rad),
The IgG10 ~ was added to 1 me sodium carbonate^am solution (
It was dissolved in 0.01M PI (9,5>) and dialyzed against the same buffer at 4°C for 20 hours, then mixed with the activated peroxidase solution and reacted at room temperature for 3 hours.Furthermore, it 'b To test the peroxidase and the peroxidase-labeled anti-β-Δ18P antibody, o.
o i), 4 phosphorus i? 1 solution (pal 7.4) was subjected to gel filtration, and peroxidase-labeled anti-β-MS
P antibody was obtained.

Example 7 Ig obtained from antibody-attached polystyrene ball anti-β-MS P I'll i'i'J
0.01 heart so that G becomes 17'lSl/me,
1 phosphate buffer (pit 7.4). This IITO1? To 20 vte of solution F, add ethanol and 0. OIIIJ! J N fee iR loose raccoon s N
(pH 7,4) 50 washed sita polystyrene balls (1/4 inch) were immersed in the solution and reacted at 4°C for 18 hours. Then add 0.01 M phosphate buffer (pH 47,
4) Washed 4 times with 1% bovine albumin (manufactured by Sigma)
and 0.01M phosphorus pre-sewing solution containing 0.05% sodium nitride (PTL 7.4
) before use.

Example 8 Measurement of β-b+ S P 4q degree in serum by enzyme immunoreaction A plastic tube containing 0.1% bovine albumin (Sigma N) and 0.05% Triton-X100 (Wako Junkuwa Co., Ltd.) .01 M phosphorus [: Seishio gauze 11i liquid Q
, 5 * (! and serum 0.1 rn, e added 11')
After stirring, add one polystyrene ball to the antibody I'Iff and react at 37°C for 1 hour.The polystyrene ball is then washed with the above PA'9h solution, and then added with 0.5 m and 0.5 m of the above buffer solution. Vermexidase labeled anti-β-MS
Add P antibody 0.11+4 and react at 37°C for 1 hour. Next, after washing the polystyrene ball with the above buffer, it was placed in another Plusdec tube and 0. 0.05 M phosphate weak fif containing 15O-7 22-diamine W salt
fi liquid (pH 7,2) 05 ytl! and 0.003%
Add hydrogen peroxide solution (157) and react at 37°C for 1 hour.
After stopping the reaction by adding H1, the absorbance at 492nrn is measured in a separate room, and the β-MSP concentration is determined from the 3-weight cotton.

[Brief explanation of the drawing]

Figure 1 shows serum PAP and β-
Ms p (7) g: Shows the results of measuring the degree. Figure I) Does not indicate the upper limit of the positive value of A P. Also, ■ does not indicate the upper limit of the normal value of β-M 8 P. 1゛, "r ii'l'outpfi'j person Hara 3 part (1 person) Engraving of the drawing (no change in content) Flute -Diagram・-m-Maedatetsugeshugetsukibadairuto, Punishment Conspiracy A Patient (Hakugo Tsuri) Procedural Amendment IF (Method) December 1982) Director General of the Japan Patent Office Kazuo Wakasugi 1, Indication of Case 1982 Patent Application No. 147731 2. Name of the invention Composition for diagnosing prostate diseases 3. Relationship with the case of the person making the amendment Patent applicant: Mibu Fu, 34-6-30 Dazaifu, Dazaifu City, Fukuoka Prefecture (and 1 other person) 4. Agent; σ〉1-1 Co., Ltd., November 12, 1982 (Delivery date: November 30, 1981) As shown in Drawing 7 and the contents of the amendment attached.

Claims (2)

[Claims] (1) A composition for diagnosing prostate disease, which comprises at least an anti-β-Mf9r antibody. (2) The anti-β-M8P antibody is labeled with a labeling substance for radioimmunoassay, f'l\'N immunoassay, or photoimmunoassay. Compositions as described.