JPS63101331A - Antigen for inducing production of human sperm immobilizing antibody - Google Patents

Abstract

PURPOSE:To obtain the titled antigen, consisting of a sperm coating antigen, secreted from male accessory genital organs and adhering to sperms and usable for contraceptive vaccines, etc. CONSTITUTION:An antigen, obtained by subjecting human seminal plasma which is optionally treated with trypsin to gel filtration chromatography with SEPHACRYL S-300(R), having about 20,000mol.wt. (present in the seminal plasma as a high polymer having >=440,000mol.wt.), reducing the antigenic activity by alkali treatment with 0.05 M NaOH (pH 13) at 20 deg.C for 1hr, heat treatment at 100 deg.C for 10min, oxidation treatment with a periodate in 10mM concentration at 20 deg.C for 1hr or treatment with a protease mixture produced by Streptomyces griseus without substatially changing activity by acid treatment with 0.1 M glycine-HCl (pH 2.5), reduction treatment with 1 M 2-mercaptoethanol, treatment with tryupsin and treatment with mixed glycosidase and capable of inducing the production of human sperm immobilizing antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a novel antigen capable of inducing the production of human sperm immobilizing antibodies. This antigen has use as a contraceptive vaccine and reagent.

[Prior Art] It has been reported that sperm immobilization antibodies exist in more than ten percent of women with unexplained infertility. However, research on the antigens to which these sperm immobilization antibodies correspond has not progressed.

[Problems to be Solved by the Invention] The purpose of the present invention is to provide a novel antigen that can induce the production of human sperm immobilization antibodies.

[Means for Solving the Problems] That is, the present invention is directed to sperm coating antigens that are secreted from the male genitalia and adhere to spermatozoa.
ntigen), which has a molecular weight of about zooooo, and is present in seminal plasma as a polymer with a molecular weight of about 440,000 or more, and is a protease mixture produced by alkali treatment, heat treatment, oxidation treatment, or Streptomyces griseus. Provides an antigen whose antigenic activity is reduced by treatment with , whose antigenic activity is not substantially changed by acid treatment, reduction treatment, trypsin treatment, or mixed glycosidase treatment, and which can induce the production of human sperm immobilization antibodies. do.

[5? :Effect of Koro I] The present invention provides a novel human sperm immobilization antibody production-inducing antigen. The antibody corresponding to the antigen of this invention has a very large sperm immobilization region, as described below. Therefore,
The antigen of this invention can be used as an excellent contraceptive vaccine. Furthermore, human sperm immobilizing antibodies corresponding to the antigens of this invention can be used as contraceptives by being applied topically to the genitals together with complement. It also has use as a reagent for inducing the production or purifying .

[Detailed Description of the Invention] The inventor of the present application has previously filed Japanese Patent Application No. 153581/1986.
The present invention, which describes a high-titer human sperm-immobilized human monoclonal antibody and a bilitoma producing it, relates to the corresponding antigen of such a human sperm-immobilized human antibody. ''; Hybrid 1-1 which produces human sperm immobilizing human monoclonal antibodies stimulates peripheral blood lymphocytes of infertile women with sperm immobilizing antibodies with PWM lectin and human sperm, and stimulates them according to a conventional method. Obtained by fusion with mouse myeloma cells. The human sperm immobilization human monoclonal antibody produced by the obtained bilitoma belongs to the human immunochlorin M group, and has a sperm immobilization value of approximately 5000 or more based on Si2°, and a sperm agglutination value of approximately 5,000 or more. It has a dilution of 1:1600 or more and specifically reacts with human ejaculated sperm, seminal plasma and sperm, human breast milk, human serum, human fumaru extract, human liver extract, human spleen extract, human brain extract. It does not react with fluid, human red blood cells, or human white blood cells, and shows weak cross-reactivity with lid ejaculated sperm, but it does not react with bovine ejaculated sperm, goat ejaculated sperm, rabbit ejaculated sperm, single sperm in hamster testicles, and sperm on the testis of worms. It does not react with body sperm.

When this Sakae-clone antibody was further subjected to an antibody absorption test, the antibody activity was almost completely absorbed in seminal plasma of an azoospermia patient (0 mg/ml) and seminal vesicle gland secretion (10 II g/ml). In addition, the prostate extract is 50B/ml and 4H
Whether the activity or absorption of Fumaru extract is 100mg/■
1 was not active or absorbed. Based on these basics, the corresponding antigen of the human sperm immobilized human monoclonal antibody is:
It was thought to be a so-called sperm coat antigen, which is secreted from the male genitals and attaches to sperm.

Therefore, human seminal plasma was fractionated by gel filtration chromatography, and each fraction was analyzed by enzyme immunoassay using the above-mentioned Ton-clone antibody as an adsorbent. Admitted. From this fraction,
The corresponding antigen of the human sperm immobilized human monoclonal antibody was successfully isolated by SDS polyacrylamide gel electrophoresis followed by Western blotting.

The human sperm immobilization antibody production-inducing antigen of this invention is 0,
05 M Na OIf (p II ], 3)
Alkali treatment (20'C, 1 hour), 100
℃ for 10 minutes, oxidative treatment with periodate at 10 μM (20 °C for 11 hours), or treatment with a protease mixture (trade name Pronase) produced by Streptomyces griseus (concentration 0.1 H). /m
1 hour at 37°C) reduces its antigenic activity.

However, 0.1 M glycine-IIC: l
(pH 2,5) (20°C, 11 maki ma), reduction treatment with 2-mercaptoethanol at a concentration of IM (2.
Treatment with trypsin at a concentration of 0.1 B/ml (37°C, 1 hour) and treatment with glycosidase at a concentration of 1g/l (37°C, 1 hour) substantially reduced the antigenic activity. does not change. Note that each process in the claims means a process under the above conditions.

The antigen of this invention has use as a contraceptive vaccine since it induces the production of human antibodies that immobilize human sperm. Furthermore, since it has the ability to specifically bind to human sperm-immobilized human antibodies, it can be used as a reagent for purifying the antibodies that can be used as contraceptives.

[Example] Peripheral blood lymphocytes were collected from a 39-year-old woman who had been infertile for over 15 years and had strong sperm immobilization and sperm agglutination antibodies. Routine medical examinations of this woman revealed no other reasonable cause of infertility, except for the presence of high titers of sperm immobilization and sperm agglutinating antibodies in the serum.

The antibody titer in her serum is the sperm immobilization value or SI 5
．． Tojite 45.0 (Isoshima and Kayama, QuanLita
tive esti+5ation of super
m immobilizing ar+Libod
y in the 5 era of woman
with 5territy of unknown
etiology: the 50% sperm i
mmmobilization unit (S15o)
.

In Recent eight advances in
Truman Reproduction (Cam
pos daPaz,, A., Drill, V.
A., 1lyashi.

11,, Redrigues, W., and 5c.
Hally, A.V., eds).

pplO-15, ExcerptaMedica, A
msterdam; 1976), sperm agglutination value or 1 = 3
2 dilution (Friberg, J. (+974).

8 simple and sensitive micro+
iethodfordemonstration o
f sperm-agglutinating act
ivity in serum fromminfert
ile @en and women, Acta 0
bsteL, Gynecol, 5cand, 5up
pl.

36:2l-29). Peripheral blood lymphocytes were isolated by Fico-Conley density gradient centrifugation.

Lymphocyte stimulation PWM lectin (final concentration 1:100, manufactured by Gibco, USA)
and 10% fetal bovine serum (Fe2).
1. Place the collected lymphocytes in PMI 1640 medium. Ox
10' and 5 x IG' washed sperm from a healthy person
and suspended. This mixture was dispensed into 15 wells of a Corning plate (Corning, USA, Corning-25820) and incubated in a 51 GO2 incubator for 3
The cells were cultured at 7°C for 5 days. After culture, stimulated lymphocytes collected from individual wells were pooled, washed twice, and
Suspended in 0ml RPMI 1640 medium.

After this stimulation operation, 58% of the lymphocytes were found to be alive when examined by trypan blue staining.

Cell fusion Shigeta et al.'s method (Sperm-immobilizing
i+onoclonalantibody to
human seminal plasma
antigens.

Cl1n, Exp, 1m5uno1.42:45
8-462 (1980)), in the presence of polyethylene glycol with a molecular weight of 100, the previously obtained stimulated live lymphocytes 5.3 x In' and the same number of mouse myeloma cells (P: 1 -N5-1/1.A
g, 4: fused with N5-1). Place the fused cells of IO in each of the 96 wells and sz co
The cells were cultured at 37°C in an incubator. 27 of the 96 wells contained only 1 x 10' lymphocytes per well, and the other 69 wells contained 4 x 10' lymphocytes per well plus 6 as a feeder layer. × 10′ mouse thymocytes were housed. After 2-4 hours of culture, change to HAT selection medium and culture for 12 hours.
．． 19. and 230 days later, the supernatants of individual wells were tested for production of human immunoglobulin by the sandwich enzyme immunoassay described below. After 23 days of culture, 14 out of 27 wells without feeder layer and 44 out of 69 wells with feeder layer.
Cell proliferation was observed in the wells.

Detection of human immunoglobulins by enzyme immunoassay All reagents used in the sandwich enzyme immunoassay were from Cavell Laboratories, USA. Polyvinyl plate (
USA, )・Arco: / -3912)17) 9 '6
Quantity (') 0.05M weight i5e Mi
'ia Rabbit anti-human immunoglobulin (IgG + 1gA÷IgM) antiserum IgG fraction in buffer (pH 9,6) (protein concentration 0.8μg/100 pLl/well)
and cultured overnight at 4°C. The unbound areas of the plate were filled with 0.15 M phosphate buffer (pH 7,
4) Blocked by adding (blocking solution) to the wells and incubating for 1 hour at room temperature.

After washing the wells with phosphate buffer containing 0.05X Tween 20, 50 ml of culture supernatant was added and cultured at room temperature for 2 hours. Giant ab') 2 made from goat anti-human immunoglobulin serum was labeled with peroxidase and diluted to 1:4000 with blocking solution.
#L+ was added to the wells. After incubating for 1 hour at room temperature, the plate was washed and diluted with orthophenylenediamine (manufactured by Wako Tsumugi Kogyo Co., Ltd.) in IM citrate M buffer (pt + 5.0) containing 0.0052) 120t at a final concentration of o.
ozx. The reaction was 50μI at 10$H.
□The reaction was stopped by adding SO4 and measured using a microplate photometer (MTP 12, manufactured by Corona Electric Co., Ltd.). As a result, among the 58 wells in which proliferation was observed,
Human immunoglobulin was detected in 22 wells.

Detection of anti-sperm antibodies by enzyme immunoassay 96-well polyvinyl plate (Falcon: 19
12) was coach inked with washed human sperm. Coach ink is a 50 locus 1 sperm suspension (6x10'/ml)
by adding to individual wells and air drying at room temperature. Plates were stored at 4°C until use.

Phosphate buffer containing 12 BSA and 2z normal horse serum was added to individual wells and kept at room temperature for 1 hour to flock the protein binding regions of the wells. After washing with phosphate buffer, 50 wells of culture supernatant were added to the sperm coated wells and incubated for 1 hour at room temperature. Thereafter, anti-human sperm antibodies were detected in exactly the same manner as the immunoglobulin detection described above. As a result, anti-human sperm antibodies were detected in 1 well out of 22 wells in which human immunocropurin was detected.

Cloning of anti-human sperm antibody-producing cells. Hybridomas in wells in which anti-human sperm antibodies were detected were incubated at 15% F.
6 x 10' in RPMI 1640 medium containing e2
/well of mouse thymocytes at 37°C.
□Cultured in incubator. Cultivation was carried out by filling 8 wells in the first row of a 96-well Konink plastic plate with the above culture medium at a concentration of approximately 1 x 10' hybridoma/well, and serially diluting each row at 1:2. The last column was therefore diluted 1:20. Culture 1
After 7 days, cell proliferation was observed in 4 of the 96 wells, and strong anti-human sperm antibodies were detected in 2 of them. A hybridoma was collected from one of the wells, and a second round of cloning was performed in exactly the same manner as the previous one. As a result, cell proliferation was observed in 46 out of 96 wells, of which 42
Production of anti-human sperm antibodies was observed in some wells. Among the 42 wells, the well with the highest anti-human sperm antibody titer,
A hybridoma was collected from the cell, and a third cloning was performed in the same manner. As a result, cell proliferation was observed in 56 out of 96 wells, and strong anti-human sperm antibodies were detected in all 56 wells. One of these wells was taken and used for the following experiment.

This Hyturitoma was transferred to a 24-well plastic plate (Corning-25820) and incubated at 37°C for 5 days.
Cultured in Co2 incubation. After the cells have grown, the culture is transferred to a tissue culture flask (Corning-25100).
and 25110) and cultured. Human sperm immobilized human monoclonal antibodies were collected from this culture.

Antibody Absorption Test An antibody absorption test was conducted to examine the specificity of this human sperm immobilized human single clone antibody.

Antibody absorption tests use various tissue extracts (homogenate supernatants), body fluids, and sperm from various animals as immunoabsorbers to absorb the culture supernatant of this hybridoma, and the remaining antibody activity is measured in a sperm immobilization test. This was done by asking for it. One well of culture supernatant appropriately diluted with a 10z saline solution of normal human serum was mixed with 100 l of serially diluted immunoabsorber, and the mixture was incubated overnight at 4°C. Before the test, the antibody activity of the culture supernatant was measured with Sl.
,. The value was adjusted to about 10 units. After incubation, the supernatant was centrifuged at 9500 g for 10 minutes) and the sperm immobilization value was examined. human semen (RAS), human breast milk (IIM)
) and normal human serum (NH3) were precipitated with ammonium sulfate, extensively dialyzed against distilled water, and lyophilized.

The body fluid stored in the seminal vesicles was similarly dialyzed and freeze-dried. For the immunoabsorption test, these were dissolved in physiological saline. The animal sperm was washed three times with physiological saline and then used for the immunoabsorption test. The results are shown in Table 1.

Table 1 +16-: Characteristics of lc4 single clone antibody Immunoglobulin class IgM Biological activity Sperm immobilization + (5000515o) Sperm aggregation + (1:160G dilution) Specificity for human tissues Ejaculated sperm 10 Breast milk proteins − Serum proteins − Jidai − Epididymis Undetermined seminal vesicle storage fluid 10 Prostate Liver − Lungs − Brain − Type B red blood cells − White blood cells − Azoospermia patient seminal plasma Deca seminal vesicle gland secretion 10 These The results show that the corresponding antigen of the human sperm immobilized human monoclonal antibody is the so-called sperm coat antigen, which is secreted from the male accessory genitalia and attaches to sperm.
ng antigen).

In addition, the species specificity (reactivity to sperm) of the single clone antibody is as follows.

Pig Cow - Goat - Rabbit - Hamster - Rat - +1. Preparation of corresponding antigen of human sperm immobilized human monoclonal antibody ゛Human seminal plasma and trypsinized human seminal plasma were mixed with Sephacryl S-:100.
(trade name) was subjected to gel filtration chromatography. Gel filtration chromatography uses a column size of 1.5
x 90cm column, phosphate buffer (pl-17,4) was used as the eluent, and the eluate flow rate was
/ hour, and fractions of 2 ml/tube were collected. As a sample, 10 g of human seminal plasma (saturated ammonium sulfate precipitated fraction)
11 samples were each subjected to chromatography using 1.5 g/ml, or a mixture prepared by adding trypsin (1 g/sl) and reacting at 37° C. for 1 hour. For each fraction obtained by this gel filtration chromatography, using the human sperm immobilized human single clone antibody as an immunoadsorbent,
Enzyme immunoassay using competition with human sperm was performed.

This enzyme immunoassay was performed using plates coated with human washed sperm (3 x In'/well). After blocking, bilidoma culture supernatant diluted 20 times (50
gl/well) and gel filtration each fraction (50,1/well)
was added and reacted overnight at 4°C. Thereafter, the method described in Detection of anti-sperm antibodies by enzyme immunoassay was performed.

The results of this enzyme immunoassay are shown in FIG. In FIG. 1, triangles and black triangles show the results for human seminal plasma, and circles and black circles show the results for human seminal plasma treated with trypsin.

As shown in FIG. 1, the fraction whose binding to human sperm immobilized human monoclonal antibodies is inhibited by human sperm, ie.

The fraction having antigenic activity against this antibody has a molecular weight of approximately 44
It was found that there were more than 10,000 polymer fractions.

This high molecular fraction (human seminal plasma) with a molecular weight of approximately 440,000 or more is then subjected to SDS polyacrylamide gel electrophoresis, and further Western blotting using the human sperm immobilized human single clone antibody described above as an immunosorbent. I did it. These were performed as follows. Laem+wi
SDS polyacrylamide gel electrophoresis was performed by the method of i et al. The first peak of the gel filtration chromatography (antigen fraction) was divided into 57.5% per lane. g (amount of protein). A 14% gel was used. Western Flotte ink was prepared using 25mM Tristlcl (
p118. ])-192 + sM glycine/2 (]1% methanol) and applying a current of 60 volts for 2 hours. For antigen detection on nitrocellulose, the IgM fraction purified by gel filtration from a serum-free medium was treated with biotin. Using a labeled humanized sperm immobilization monoclonal antibody, avidin-
Biotin-peroxidase complex (VEC-ASTA
! N), Next time 0.05$ 4-'y mouth ai-+7toll and (1,01$H20□-TBS were reacted.(
TBS: 50mM Tris lH: l-200mM
NaCl pH 7,4) was reacted. As a result, a molecule with a molecular weight of about 20,000 that specifically binds to the human sperm immobilized human single clone antibody was isolated.

From the above results, the antigen of the present invention, which corresponds to the human sperm-immobilized human antibody, has a molecular weight of approximately 20,000 molecule-1 and is present in seminal plasma as a polymer with a molecular weight of approximately 440,000 or more. It became clear.

111. Analysis of antigen Seminal plasma (IB/+*l) containing the above antigen was subjected to various treatments shown in Table 2. Thereafter, an enzyme immunoassay similar to that described above was performed to examine changes in antigen activity. The results are shown in the same table. Furthermore, the results of enzyme immunoassay when treated with various enzymes or oxidizing agents are shown in FIGS. 2 and 3. In FIG. 2, black circles show the results of the control (phosphate buffer), black triangles show the results of treatment with pronase (trade name), white circles show the results of treatment with tripridine, and white triangles show the results of treatment with glycosidase. In Figure 3, the white circles are the control (phosphate buffer);
The black circles indicate the results when treated with periodic acid.

As can be seen from the above results in Table 2, the antigen activity of the present invention decreased with pronase (trade name), did not change with mixed glycosidase, and as shown in FIG.
It is considered to be a glycoprotein since it has absorption in 8-on-Peng.

[Brief explanation of the drawing]

Figure 1 shows the results of gel filtration chromatography and enzyme immunoassay of human seminal plasma and trypsin-treated human seminal plasma. Figures 2 and 3 show the antigen activity of the antigen of this invention after various treatments. It is a graph showing changes in.

Claims (2)

[Claims] (1) Sperm coat antigen secreted from the male accessory genitalia and attached to sperm, with a molecular weight of approximately 20,000, and present in seminal plasma as a polymer with a molecular weight of approximately 440,000 or more. Immotile human spermatozoa whose antigenic activity is reduced by treatment or treatment with a protease mixture produced by Streptomyces griseus and whose antigenic activity remains substantially unchanged by acid treatment, reduction treatment, trypsin treatment, mixed glycosidase treatment. Antigens that can induce the production of antibodies. (2) the human sperm immobilizing antibody is produced by a hybridoma obtained by fusing mouse myeloma cells with lymphocytes of an infertile woman carrying the sperm immobilizing antibody;
It belongs to human immunoglobulin M having a λ chain, has a sperm immobilization value of about 5000 or more as Si_5_0, a sperm agglutination value of about 1:1600 dilution or more, and human ejaculated sperm,
Reacts specifically with seminal plasma and seminal vesicles, and reacts with human breast milk, human serum, human testicle extract, human liver extract, human spleen extract, human brain extract, type B human red blood cells, and human white blood cells. immobilized human single clone that shows weak cross-reactivity with pig ejaculated sperm but does not react with bovine ejaculate, goat ejaculate, rabbit ejaculate, hamster epididymal sperm, and rat epididymal sperm. The antigen according to claim 1, which is an antibody.