JP2534480B2 - Corresponding antigen of human sperm immobilization antibody - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel corresponding antigen of human sperm-immobilized antibody. This antigen has uses as a contraceptive vaccine and reagent.

[Prior Art] Conventionally, the presence of sperm-immobilizing antibodies has been reported in more than 10% of infertile women of unknown cause. However, studies have not been conducted on the corresponding antigens of these sperm-immobilized antibodies.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel corresponding antigen for human sperm-immobilized antibody.

[Means for Solving Problems] That is, the present invention is directed to a sperm coating antigen secreted from male accessory organs and attached to sperm.
, Whose molecular weight is 20000, exists as a polymer having a molecular weight of 440,000 or more in seminal plasma, and the antigen is treated by alkali treatment, heat treatment, oxidation treatment or treatment with a protease mixture produced by Streptomyces griseus. Provided is a corresponding antigen of a human sperm-immobilized antibody whose activity is reduced and whose antigen activity is not changed by acid treatment, reduction treatment, trypsin treatment, and mixed glycosidase treatment.

EFFECTS OF THE INVENTION The present invention provides a novel corresponding antigen for human sperm-immobilized antibody. The antibody against the antigen of the present invention has a very large sperm immobilization ability, as described later. Therefore,
The antigen of this invention can be used as an excellent contraceptive vaccine. Furthermore, the human sperm-immobilizing antibody corresponding to the antigen of the present invention can be used as a contraceptive by locally applying to the genitals together with complement, but the antigen of the present invention is It also has uses as a reagent for inducing or purifying production.

[Detailed Description of the Invention] The inventor of the present invention described in Japanese Patent Application No. 61-153581 filed previously, a high-titer human sperm-immobilized human monoclonal antibody and a hybridoma producing the same. The present invention relates to such a corresponding antigen of a human sperm-immobilized human antibody. The hybridoma producing the human sperm-immobilized human monoclonal antibody is a peripheral blood lymphocyte of an infertile woman having a sperm-immobilized antibody, which is stimulated with a PWM lectin and human sperm, and fused with mouse myeloma cells according to a conventional method. Obtained by The human sperm-immobilized human monoclonal antibody produced by the obtained hybridoma belongs to human immunoglobulin M having a λ chain, has a sperm immobilization value as SI ₅₀ of about 5,000 or more, and a sperm aggregation value of about
1: 1600 dilution or more, reacts specifically with human ejaculate sperm, seminal plasma and seminal vesicles, human breast milk, human serum, human testis extract, human liver extract, human spleen extract, human brain extract It does not react with liquid, human erythrocyte, human leukocyte, and shows weak cross-reactivity with porcine ejaculate sperm, but with bovine ejaculate, goat ejaculate, rabbit ejaculate, hamster epididymal sperm and rat epididymal sperm no response.

When an antibody absorption test was further carried out on this monoclonal antibody, the antibody activity was almost completely absorbed in 1.0 mg / ml seminal plasma of a patient with azoospermia and 10 mg / ml of seminal vesicle secretory fluid. Furthermore, 40% of the activity was absorbed in the prostate extract at 50 mg / ml, but not at 100 mg / ml in the testis extract. From these facts, the corresponding antigen of the human sperm-immobilized human monoclonal antibody was considered to be a so-called sperm-covering antigen that is secreted by male sex organs and attached to sperm.

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

The corresponding antigen of the human sperm-immobilized antibody of the present invention is 0.05M.
Alkaline treatment with NaOH (pH13) (20 ℃, 1 hour), 10
Heat treatment at 0 ° C for 10 minutes, oxidation treatment with a concentration of 10 mM periodate (20 ° C, 1 hour) or treatment with a protease mixture (trade name Pronase) produced by Streptomyces griseus (concentration 0.1 mg / ml) , 37 ° C, 1 hour), its antigenic activity decreases. However,
Acid treatment with 0.1 M glycine-HCl (pH 2.5) (20 ° C, 1 hour), reduction treatment with 1M concentration of 2-mercaptoethanol (20 ° C, 1 hour), treatment with 0.1 mg / ml concentration of trypsin (37 The antigenic activity is not substantially changed by treatment with glycosidase at a concentration of 1 mg / ml (37 ° C, 1 hour). Each process in the claims means a process under the above conditions.

Since the antigen of the present invention is considered to be capable of inducing the production of human sperm-immobilized human antibody, it is considered to have use as a contraceptive vaccine. Further, since it has the ability to specifically bind to human sperm-immobilized human antibody, it has a use as a reagent when purifying the antibody that can be used as a contraceptive.

[Examples] I. Preparation of human sperm-immobilized human monoclonal antibody Lymphocyte collection Peripheral blood lymphocytes were collected from a 39-year-old female who was infertile for 15 years or more and had a strong sperm immobilization and sperm aggregation antibody. When this woman was examined by routine medical examination, no other reason for infertility was found, except for the presence of high titers of sperm immobilization and sperm agglutination antibodies in the serum. The antibody titer in her serum was 4 with a sperm immobilization value of SI _50.
5.0 (Isoshima and Kayama, Quantitative estimation of sperm
immobilizing antibody in the sera of woman with st
erility of unknown etiology: the 50% sperm immobil
ization unit (SI ₅₀ ) .In Recent Advances in Human R
eproduction (Campos daPaz ,, A., Drill, VA, Hyashi,
H., Redrigues, W.and Schally, AVeds), pp10-15, Excer
ptaMedica, Amsterdam; 1976), sperm aggregation value diluted 1:32 (Friberg, J. (1974). A simple and sensitive microm
ethod for demonstration of sperm-agglutinating act
Activity in serum from infertile men and women.Acta Ob
stet.Gynecol.Scand.Suppl.36: 21-29). Peripheral blood lymphocytes were isolated by Ficoll-Conley density gradient centrifugation.

Stimulation of lymphocytes PWM lectin (final concentration 1: 100, US Gibco) and 10%
In 15 ml RPMI 1640 medium containing fetal calf serum (FCS),
1.0 × 10 ⁷ collected lymphocytes and 5 × 10 ⁶ washed sperm from a healthy person were suspended. This mixture was added to a Corning plate (Corning USA, Corning-25820) 1
Dispense into 5 wells, 37 ℃ in 5% CO ₂ incubator
The cells were cultured for 5 days. After culturing, stimulated lymphocytes collected from individual wells were pooled, washed twice and 10 ml RP
Suspended in MI 1640 medium. After the stimulation operation, 58% of the lymphocytes were found to be alive when examined by the trypan blue staining method.

Cell fusion Shigeta's method (Sperm-immobilizing monoclonal anti
body to human seminal plasma antigens.Clin.Exp.Imm
unol.42: 458-462 (1980)), in the presence of polyethylene glycol having a molecular weight of 1000, 5.3x10 ⁶ stimulated living lymphocytes obtained previously and the same number of mouse myeloma cells (P3- NS-1 / 1.Ag.4: NS-1). 1
Add 00 μl of fused cells to each of 96 wells, 5
Incubated at 37 ° C in a% CO ₂ incubator. Twenty-seven of the 96 wells contained only 1x10 ⁵ lymphocytes per well and the other 69 wells contained 4x10 ⁴ per well.
And ⁶ × 10 ⁵ mouse thymocytes as feeder layers. After 24 hours of culture, the medium was changed to a HAT selection medium, and after 12, 19, and 23 days of culture, the supernatants of individual wells were tested by the sandwich enzyme immunoassay described below to examine whether they produced human immunoglobulin. . culture
After 23 days, cell proliferation was observed in 14 of 27 wells without the feeder layer and in 44 of 69 wells with the feeder layer.

Detection of Human Immunoglobulins by Enzyme Immunoassay All reagents used for sandwich enzyme immunoassay were from Capell Laboratories, USA. 96 wells of a polyvinyl plate (Falcon-3912, USA) were loaded with rabbit anti-human immunoglobulin (IgG + IgA + IgM) antiserum IgG fraction (protein concentration 0.8 μg / 100 μl / well) in 0.05 M bicarbonate buffer (pH 9.6). ) And cultured overnight at 4 ° C. The unbound area of the plate was prepared by adding 0.15 M phosphate buffer (pH 7.4) (blocking solution) containing 1% bovine serum albumin (BSA) and 5% normal horse serum to the wells and incubating at room temperature for 1 hour. Blocked by. After washing the wells with a phosphate buffer containing 0.05% Tween 20, 50 μl of culture supernatant was added, and the cells were cultured at room temperature for 2 hours. F made from goat anti-human immunoglobulin serum
50 μl of (ab ′) ₂ labeled with peroxidase diluted 1: 4000 with blocking solution was added to the wells. After incubating for 1 hour at room temperature, wash the plate, and add orthophenylenediamine (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.02% in 0.1M citrate buffer (pH 5.0) containing 0.005% H ₂ O _2. Added to. The reaction is 50 μl of 10% H ₂ SO
_It was stopped by adding ₄ and measured with a microplate photometer (MTP 12, manufactured by Corona Electric Co., Ltd.). As a result, human immunoglobulin was detected in 22 of the 58 wells in which proliferation was observed.

Detection of anti-sperm antibody by enzyme immunoassay 96-well polyvinyl plate (Falcon-3912)
Were coated with washed human sperm. Coating was done by adding 50 μl of sperm suspension ( ⁶ × 10 ⁶ / ml) to each well and air drying at room temperature. Plates were stored at 4 ° C until use. Phosphate buffer containing 1% BSA and 2% normal horse serum was added to each well,
The protein binding regions of the wells were blocked by holding at room temperature for 1 hour. After washing with phosphate buffer, 50 μl of culture supernatant was added to the sperm-coated wells and incubated for 1 hour at room temperature. Then, anti-human sperm antibody was detected in exactly the same manner as the above-mentioned detection of immunoglobulin. As a result, anti-human sperm antibody was detected in one of the 22 wells in which human immunoglobulin was detected.

Cloning of anti-human sperm antibody-producing cells Hybridomas in the wells in which anti-human sperm antibody was detected were incubated in RPMI 1640 medium containing 15% FCS with 6x10 ⁵ / well mouse thymocytes at 37 ° C for 5% CO ₂ incubation. It was cultured in. The culture was carried out by placing the above culture solution at a concentration of about 1 × 10 ³ hybridomas / well in 8 wells of the first row of a 96-well Corning plastic plate, and per row
It carried out by serially diluting 1: 2. Therefore the last column was diluted 1: 2 ¹¹ . After 17 days of culture, cell proliferation was observed in 4 of 96 wells, and strong anti-human sperm antibody was detected in 2 of them. Hybridomas were harvested from one of the wells and cloned a second time, exactly as before. As a result, cell proliferation was observed in 46 of 96 wells, and production of anti-human sperm antibody was observed in 42 of them. The hybridoma was collected from the well having the highest anti-human sperm antibody titer out of 42 wells, and the third cloning was performed in the same manner. As a result, cell growth was observed in 56 of 96 wells, and strong anti-human sperm antibody was detected in all of 56 wells. One of these wells was taken and used for the following experiments.

This hybridoma was transferred to a 24-well plastic plate (Corning-25820) and cultured at 37 ° C. in a 5% CO ₂ incubation. After the cells had grown, the culture was expanded into tissue culture flasks (Corning-25100 and 25110) and cultured. Human sperm-immobilized human monoclonal antibody was harvested from this culture.

Antibody Absorption Test An antibody absorption test was performed to examine the specificity of this human sperm-immobilized human monoclonal antibody. The antibody absorption test is a sperm immobilization test for the antibody activity of various tissue extracts (homogenate supernatants), body fluids, and spermatozoa of various animals used as immunoabsorbers to absorb the culture supernatant of this hybridoma and to migrate. It was done by asking by. Culture supernatant 10 appropriately diluted with 10% normal human serum saline solution
μl was mixed with 100 μl of serially diluted immunosorbent,
This mixture was incubated overnight at 4 ° C. Prior to the test, the antibody activity of the culture supernatant was adjusted to about 10 units by SI ₅₀ value. After incubation, centrifuge the supernatant (9500g, 30
Min), and the sperm immobilization value was examined. Human semen (HAS), human breast milk (HM) and normal human serum (NHS) were precipitated with ammonium sulfate, dialyzed thoroughly against distilled water and freeze-dried. Similarly, the body fluid stored in the seminal vesicle was dialyzed and freeze-dried. In the immunoabsorption test, these were dissolved in physiological saline. The sperm of the animal was washed three times with physiological saline and then used for the immunoabsorption test. The results are shown in Table 1.

Table 1 Characteristics of H6-3C4 monoclonal antibody Immunoglobulin class IgM (λ) Biological activity Sperm immobilization ＋ (5000 SI ₅₀ ) Sperm aggregation ＋ (1: 1600 dilution) Specificity to human tissue Ejaculate sperm ＋ sperm + Breast milk protein − Serum protein − Testis − Epididymal seminal vesicle storage fluid + Prostate ± Liver − Spleen − Brain − B type red blood cells − White blood cells − Azoospermia patient seminal plasma + seminal vesicle secretory fluid + From these results The corresponding antigen of the human sperm-immobilized human monoclonal antibody is a so-called sperm coating antigen that is secreted by male accessory organs and adheres to sperm.
n).

The species specificity (reactivity to sperm) of the above monoclonal antibody is as follows.

Pig ± Bovine-Goat-Rabbit-Hamster-Rat-II. Preparation of the corresponding antigen for human sperm-immobilized human monoclonal antibody Human seminal plasma and trypsin-treated human seminal plasma are subjected to gel filtration with Sephacryl S-300 (trade name). Chromatographed. For gel filtration chromatography, use a column with a column size of 1.5x90 cm, use phosphate buffer (pH 7.4) as the eluent, and set the flow rate of the eluent to 10 ml / hour to collect 2 ml / fraction of each test tube. It was As a sample, 10 mg / ml of human seminal plasma (saturated ammonium sulfate precipitation fraction) or trypsin (1 mg / ml) added thereto and reacted at 37 ° C. for 1 hour was used, and 1 ml of each was subjected to chromatography. Each fraction obtained by this gel filtration chromatography was subjected to enzyme immunoassay using competition with human sperm using the human sperm-immobilized human monoclonal antibody as an immunosorbent. This enzyme immunoassay was performed using a plate coated with human washed sperm (3 × 10 ⁵ / well). After blocking, 20-fold diluted hybridoma culture supernatant (50 μl / well) and each gel filtration fraction (50 μl / well) were added and reacted overnight at 4 ° C. After that, the method described in the detection of anti-sperm antibody by enzyme immunoassay was performed.

The results of this enzyme immunoassay are shown in FIG. In Figure 1,
The triangles and black triangles show the results for human seminal plasma, and the circles and black circles show the results for trypsin-treated human seminal plasma. As shown in FIG. 1, the fraction in which binding to human sperm-immobilized human monoclonal antibody is inhibited by human sperm, that is, the fraction having antigenic activity against this antibody, has a high molecular weight of about 440,000 or more. It was found to be a molecular fraction.

The high molecular weight fraction (human seminal plasma) having a molecular weight of about 440,000 or more was then subjected to SDS polyacrylamide gel electrophoresis, and further the Western blotting method using the human sperm-immobilized human monoclonal antibody as an immunoadsorbent was performed. I did.
These were performed as follows. SDS polyacrylamide gel electrophoresis was performed by the method of Laemmli et al. 57 μg (protein amount) of the first peak (antigen fraction) of gel filtration chromatography was applied per lane. As the gel, 14% gel was used. Western blotting was carried out by using 25 mM Tris HCl (pH 8.3) -192 mM glycine / 20% methanol and energizing at 60 volts for 2 hours. For antigen detection on nitrocellulose, human sperm-immobilized monoclonal antibody labeled with biotin in IgM fraction purified from serum-free medium was used, and avidin-biotin-peroxidase complex (VECTASTAIN), Then 0.05% 4-chloro-1-naphthol and 0.01% H ₂ O ₂ -TBS
Was reacted. (TBS: 50 mM Tris HCl-200 mM NaCl pH 7.
4) was reacted. As a result, a molecule having a molecular weight of about 20,000 that was specifically bound to the human sperm-immobilized human monoclonal antibody was isolated.

From the above results, it is clear that the antigen of the present invention corresponding to the human sperm-immobilized human antibody has a molecular weight of about 20,000 and is present in seminal plasma as a polymer having a molecular weight of about 440,000 or more. became.

III. Analysis of Antigen The seminal plasma (1 mg / ml) containing the above antigen was subjected to each treatment shown in Table 2. Then, the same enzyme immunoassay as described above was performed to examine the change in the antigen activity. The results are shown in the table. Furthermore, FIGS. 2 and 3 show the results of enzyme immunoassay when treatment with various enzymes or oxidizing agents was performed. In FIG. 2, the black circles show the results when the control (phosphate buffer solution), the black triangles show the pronase (trade name), the white circles show the tryplysin, and the white triangles show the results when glycosidase was used. In FIG. 3, white circles show the results when the control (phosphate buffer solution) was used, and black circles show the results when the treatment with periodate was performed.

As can be seen from the above results, the antigen of the present invention has a decreased activity of pronase (trade name), is unchanged by mixed glycosidases, and as shown in FIG.
It is considered to be a glycoprotein because it has an absorption at 280 nm.

[Brief description of drawings]

FIG. 1 is a graph showing the results of gel filtration chromatography and enzyme immunoassay of human seminal plasma and trypsin-treated human seminal plasma, and FIGS. 2 and 3 show the antigenic activity of various treatments with the antigen of the present invention. It is a graph which shows the change of.

Claims (2)

(57) [Claims] 1. A sperm-coated antigen that is secreted from male genital organs and adheres to sperm, has a molecular weight of 20,000, and is present in seminal plasma as a polymer having a molecular weight of 440,000 or more, and is subjected to alkali treatment, heat treatment, and oxidation. Treatment or Streptomyces
Treatment with a protease mixture produced by Griseus reduces its antigenic activity, resulting in acid treatment, reduction treatment,
Corresponding antigen for human sperm-immobilized antibody whose antigen activity is not changed by trypsin treatment and mixed glycosidase treatment. 2. The human sperm-immobilized antibody is a human immunoglobulin M having a λ chain, which is produced by a hybridoma obtained by fusing lymphocytes of an infertile woman carrying the sperm-immobilized antibody and mouse myeloma cells. Sperm immobilization value is more than 5000 as SI ₅₀ and sperm aggregation value is 1:
1600 dilution or more, specifically reacts with human ejaculate, seminal plasma and seminal vesicles, human milk, human serum, human testis extract, human liver extract, human spleen extract, human brain extract, Human spermatozoa that do not react with type B human erythrocytes and human leukocytes and show weak cross-reactivity with porcine ejaculate sperm, but not bovine ejaculate, goat ejaculate, hamster epididymal sperm or rat epididymal sperm The antigen according to claim 1, which is an immobilized human monoclonal antibody.