Journal of Reproductive Immunology, 17 (1990) 1-16

1

Elsevier Scientific Publishers Ireland Ltd.

JR100638

Monoclonal antibody recognizing an apparent peptide epitope of human seminal plasma glycoprotein and exhibiting sperm immobilizing activity Iglika B a t o v a * ; K i n u K a m e d a , A k i k o H a s e g a w a , Y o s h i y u k i Tsuji, S h i n z o I s o j i m a

Koji K o y a m a ,

Department of Obstetrics and Gynecology, Hyogo Medical College, Nishinomiya 663 (Japan) (Accepted for publication 10 October 1989)

Summary A hybridoma (3B2-F7) has been established which secretes a monoclonal antibody (Mab) directed against a peptide determinant of human seminal plasma glycoprotein (HSP-gP). The deglycosylation of HSP-gP was performed chemically with TFMS hydrolysis and enzymatically in the presence of detergent and further treated with periodic acid after fixing deglycosylated HSP on plastic wells. The Mab 3B2-F7 (IgM, K) exhibited sperm immobilization activity (256 units of SI5o) and inhibited sperm binding to human zona pellucida. Human epididymis, pancreatic islets of Langerhan's and distal tubulus of kidney were strongly labelled whilst other tissues were essentially negative by avidin-biotin complex tissue staining with this Mab. The antigen epitope to the Mab was in the 36 kDa molecule of human HSP-gP. The antigenic determinant recognized by Mab 3B2-F7 was destroyed by six different proteases, but was resistant to N-glycanase and other carbohydrate splitting enzymes. This epitope is therefore likely to be composed of a polypeptide chain. Peptide fragments after proteolysis of the HSP molecule with Staph. aureus V8 protease and trypsin retained antigenicity, hence the epitope corresponding to the Mab may be a peptide chain and not dependent on the conformational structure of the polypeptide. Key words: monoclonal antibody; peptide epitope; human sperm immobiliz-

ation; sperm binding inhibition; antigen specificity. *Present address: Institute of Biology of Reproduction of Organisms, Sofia, Bulgaria. Correspondence to: Professor S. Isojima. 0165-0378/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

In~oducfion

Murine and human hybridomas directed to human sperm and human seminal plasma (HSP) secreting monoclonal antibodies (Mabs) with strong sperm immobilizing (SI) and agglutinating (SA) activities have been established in our laboratory for several years (Shigeta et al., 1980; Kyurkchiev et al., 1986; Isojima et al., 1987). However, the purification of the antigens corresponding to these Mabs have not been achieved (Isojima et al., 1982) and the chemical analysis of epitopes corresponding to the Mabs have been complicated. Recently, Tsuji et al. (1988) succeeded in clarifying that the chemical structure of the epitope corresponding to our human Mab H63C4 is an unbranched polylactosamine, i.e. blood group i antigen, regardless of sialic acid at the terminal galactose. We also demonstrated that many women's SI positive sera contained antibodies to carbohydrate epitopes of sperm antigens. Conversely, we recognized that some SI positive sera could be partly absorbed with deglycosylated spermatozoa. Therefore, the possible generation of antibodies to peptide epitopes of sperm antigens in women could not be ruled out. For these reasons, we aimed to establish a Mab with SI activity which reacts to the peptide portion of sperm and HSP antigens. A Mab recognizing a small linear sequential peptide epitope of sperm antigen could provide a highly specific and sensitive probe for the screening and cloning of cDNA libraries obtained from male sexual accessory glands, in order to obtain the D N A producing target antigen. Moreover, SI Mabs directed to protein moieties on sperm membrane,, including sperm coating antigens, may provide a suitable tool to analyze the location of the functional gene products which would cover the sperm during maturation of sperm cells.

Materials and methods

Production of monoclonal antibodies Balb/c mice were immunized weekly by intraperitoneal injections of human sperm (5 x 107/ml), 2-3 times. The sera were obtained one week after the final immunization and antibody titers were estimated by ELISA using plastic plates coated with human sperm or deglycosylated HSP-gp, and the titers of SA and SI antibodies were determined by our sperm immobilization test (Isojima and Koyama, 1976). Hybridomas were produced by fusing mouse myeloma cells P3x63AGU1 (P3U1) with spleen cells of the immunized mouse whose SI antibody titer was 3200 units as SIso, in the presence of PEG 4000 (Sigma). The fused cells were distributed into plastic wells (Falcon) at a concentr-

ation of 3 x 105 cells/well, kept in HAT selective medium for 14 days and transferred to HT medium. The antibody positive hybridomas were subcloned by limiting and sequential dilution methods. The selected hybridoma cells were grown in ascites form in the mouse peritoneal cavity after injection of pristane.

Deglycosylation of human seminal plasma glycoproteins by trifluoromethane sulfonic acid (TFMS) Lyophilized HSP glycoproteins (5 mg) were dissolved in 1 ml of a TFMSanisole mixture (2:1, v/v) at 0°C under nitrogen in a screw-capped tube and stirred for 2-8 h. Hydrolized carbohydrates were extracted with a 2fold volume of diethyl ether and neutralized by adding an equal volume of ice-cold 50% (v/v) aqueous pyridine. Ether extractions of the aqueous phase were repeated 3-4 times and the extract was dialysed against 2 mM pyridine acetate buffer (pH 5.5 ) and deionized water (Edge et al., 1981).

Enzymatic deglycosylation of human seminal plasma glycoproteins The following enzyme digestions were carried out sequentially: First, neuraminidase treatment of native or TFMS-deglycosylated HSP (100/zg) was carried out with 0.05 units of enzyme in 200 mM sodium acetate (pH 5.6) containing 2 mM CaClz and 1 mM phenylmethyl sulfonyl fluoride (PMSF), for 24 h at 37°C. The second, /3-N-acetylglucosaminidase (from bovine kidney, Sigma) digestion of TFMS-treated HSP glycoproteins was performed with 0.8 units of enzyme in 50 mM sodium citrate buffer (pH 5.0) for 24 h at 37°C. The enzyme was inactivated by heating. For the third, the treated sample was lyophilized and subjected to cleavage with a-N-acetylgalactosaminidase (0.01 units) in 50.mM sodium citrate (pH 4.5) containing 0.1% Triton X-100 and 1 mM PMSF, for 24 h at 37°C. The fourth,/3-galactosidase (Sigma) digestion was performed at 4 units of enzyme in 100 mM sodium phosphate buffer (pH 6.5). Endoglycosidase D (0.5 units) and glycopeptidase A (0.03 units, Seikagaku) were also used for digestion in 100 mM phosphate citrate buffer (pH 6.5) in the presence of exoglycosidase. N-glycanase (Peptide : N-glycosidase F, Genzyme, 0.03 units) was applied in 200 mM sodium phosphate (pH 8.6) in the presence of 100 mM 1,10-phenanthroline hydrate and 1.25% Nonidet P40 for 24 h at 37°C.

Enzyme-linked immunosorbent assay/ Culture supernatants were monitored for antibody production against deglycosylated HSP antigens. Microtiter plates (Falcon) were pre-treated with 0.1% BSA, followed by incubation with 0.1% glutaraldehyde in PBS for 30 min at room temperature. Deglycosylated HSP was solubilized in

50 mM Tris-HC1 (pH 7.5) containing 0.1% SDS, and 50 /xl (100 /zg of deglycosylated HSP/ml) of the solution was poured into each well of a plate. The plate was allowed to dry at 4°C and washed with PBS-containing 0.05% Tween-20 (PBS-Tween), and then rinsed with 50 mM sodium acetate buffer (pH 4.5) following incubation with 10 mM periodate in the same buffer for 1 h at room temperature. A plate was washed with PBS-Tween and incubated for 30 min with 50 mM sodium borohydride. After washing with PBS, the residual binding sites were blocked with PBS containing 1% BSA and 2% horse serum for 1 h. One hundred microliters of culture supernatants were applied to the microtiter wells and incubated for 2 h. After washing with PBS-Tween, 50 /zl (1:500) of peroxidase-conjugated goat anti-mouse Ig (Cappel) was added to the wells, incubated for 1 h and then developed by the addition of o-phenylene diamine (Wako Pure Chem., Japan) in 0.1 M citrate-phosphate buffer (pH 5.0) containing 0.005% H202. The reaction was terminated by H 2 S O 4 after 3- 5 min and the absorbance at 492 nm was measured by a microplate photometer MTP 12 (Corona Electric, Japan).

Sperm immobilization and agglutination tests The microsperm immobilization test (Isojima and Koyama, 1978) was employed. Ten microliters of the culture supernatants were added to the wells of a Terasaki plate (Falcon, U.S.A.) and overlayered with liquid paraffin oil. Two microliters of guinea pig serum as complement source and 1/zl of human sperm suspension (2 x 10 7 ml) were added under liquid paraffin oil and incubated at 34°C for 60 min. As a complement control, 2/xl of heat-inactivated (56°C for 30 min) guinea pig serum was used. The SI antibody titers were calculated as 50% sperm immobilization units (SI50) (Isojima and Koyama, 1976). Sperm agglutinations were observed in the wells containing heat-inactivated guinea pig serum after 60 min. following the same criteria as Franklin and Dukes(1964). Immunochemical tissue staining Antigen localization in tissue sections of tested tissues and human spermatozoa was performed by avidin-biotin peroxidase complex (ABC) staining (Vectastain ABC kit, Vector Lab.). Paraffin embedded tissue sections were deparaffinized by xylene and dehydrated with alcohol. The endogenous peroxidase activity was destroyed by incubation in 0.3% H202 in methanol for 30 min. After washing in 50 mM Tris-HCl buffer (pH 7.5) or PBS, tissue sections were blocked with adequately diluted normal mouse serum. After washing, the tissue sections were incubated with monoclonal antibody (culture supernatant) for 30 min and a biotinylated anti-mouse IgM (1:220) was added and incubated for 30 min. Slides were extensively washed and incubated with Vectastain ABC reagent for 40 min. The

reaction was developed with 0.1% diaminobenzidine in i00 mM Tris-HCl (pH 7.2) with the addition of H202 to a final concentration of 0.02%. After washing, slides were counterstained with Mayer's hematoxylin.

Immunofluorescence Tissue sections were deparaffinized with xylene, dehydrated with ethanol and blocked in 5% BSA-PBS. A drop of Mab (culture supernatant) was applied and maintained for 1 hr at 4°C. After washing in PBS, FITCconjugated Fab fragment of goat anti-mouse IgM (Cappel, 1:200) in PBS was applied for 30 min at room temperature. Slides were thoroughly washed and observed by immunofluorescence microscopy under one drop of glycerol. Washed fresh sperm cells were mixed with Mab culture supernatant. The sperm-Mab suspension was kept at 4°C for 30 min, centrifuged and washed in PBS. It was further treated with FITC-labelled anti-mouse IgM (1:100) in PBS for 30 min at 4°C, centrifuged and suspended in PBS after washing. A drop of this suspension on a slide was observed by fluorescence microscopy.

Proteolysis of antigen corresponding to the monoclonal antibody and immunoaffinity chromatography The antigen corresponding to the Mab was eluted from SDS-PAGE gel and subjected to proteolysis with Staph. aureus V8 protease and trypsin in 25 mM NH4HCO3 containing 0.02% SDS for 18 h at 37°C. The ratio of enzyme to substrate was 1:10. After stopping the reaction by heating at 100°C for 3 rain, the peptide mixture was applied to the Mab bound Sepharose 4B column. The culture supernatant of the hybridoma or ascites fluid was precipitated at 50% (NH4)2SO4 saturation, and the IgM fraction was separated by Sephacryl S-300 chromatography. The Mab (2 mg/ml of gel) was bound to cyanogen bromide activated Sepharose 4B column (0.5 × 1 cm) and equilibrated with PBS containing 0.5% NP-40. After alternative washings with high salt (2 M NaC !, 100 mM sodium phosphate, pH 7.4) and low pH buffers (50 mM sodium acetate, pH 4.0), the bound peptides were eluted with 0.5 M acetic acid.

SDS-PAGE and Western blotting HSP-gps were fractionated on 12% polyacrylamide gels according to Laemmli (1970). Gels were stained with 0.1% Coomassie Blue R250 in methanol-acetic acid and destained. In some experiments the gels were further stained with periodate-Schiff reagent (Fairbanks et al., 1971). Detection of antigen corresponding to the Mab was carried out by electroblotting on a nitrocellulose membrane (Towbin et al, 1979). The membrane was blocked in the presence of Tween-20 (0.05%) and reacted with Mab.

After washing in PBS-Tween the membranes were incubated with secondary Ab, goat anti-mouse IgM conjugated peroxidase. The reaction was developed in 0.1% diaminobenzidine in the presence of 0.02% H202. Results

Effects of deglycosylation on HSP molecules HSP antigens were subjected to acid methanolysis with TFMS at 0°C for various times ranging from 2 to 8 h. Figure 1 shows the time course of the effect of deglycosylation on the HSP molecules. All preparations lacked detectable low molecular weight material stainable by Coomassie Blue and periodate Schiff which might result from strong proteolysis. Reduction in the molecular size of major HSP components, which was shown by sharp stained zones, was already observed with TFMS treatment after 2 h and additional new bands appeared during the process of longer TFMS treatment up to 8 h. The PAS-stainable material of HSP glycoprotein at the

2

3

4

S

6

7 94Kd 67Kd 43Kd 30Kd 20.1Kd

14.4Kd

Fig. 1. SDS-PAGE: Time course of deglycosylation of human seminal plasma glycoprotein with trifluoromethanesulfonic acid. Lane 1, non-treated; Lane 2, 2h treatment; Lane 3, 3h treatment; Lane 4, 4h treatment; Lane 5, 5h treatment; Lane 6, 8h treatment; Lane 7, calibration kit (Lactalbumin, 14.4 kDa; trypsin inhibitor 20.1 kDa; carbonic anhydrase, 30 kDa; Ovalbumin, 43 kDa; bovine serum albumin, 67 kDa; phosphorylase b, 94 kDa).

starting level of the gel was diminished and the deglycosylated sample migrated into the running gel. Routine deglycosylation was performed with TFMS for 8 h at 0°C, for further experiments, though this condition might not be optimal to deglycosylate individual proteins because of the heterogeneity of HSP glycoproteins. To extract more carbohydrate moieties from the antigenic material, the lyophilized TFMS-treated HSP was digested with exo- and endoglycosidases and a neuraminidase was also used before or after TFMS hydrolysis. For selecting the target hybridoma which produces Mab to the peptide portion of HSP, the above enzymatic deglycosylations were applied to the TFMS-treated HSP glycoproteins before or after immobilizing them on the microtiter plates. In order to solubilize the deglycosylated HSP proteins, chaotropic agents, urea, guanidine hydrochloride or detergents were required. Therefore, to coat the deglycosylated HSP antigens effectively on a microplate, SDS, a detergent with high critical micelle concentration value (CMC), was used for solubilization at a concentration below the CMC. The effect of periodic acid (PI) treatment on bound deglycosylated HSP on a plate was examined by ELISA using anti-mouse human sperm serum (Fig. 2). Some reduction in antigenicity was observed after PI treatment. 1.5

1.0

O

0.5

0

I

I

100

I

200

400

O

I

I

800

1,600

3) 200

6,400

1/Dilution 0

: DG

~

: DG-PA

[-] : D G - P A - P r o n a s e

Fig. 2. Titration curves of antiserum by ELISA. Abscissa, dilution of antiserum; Longitude, A492. O - - O - - O , Deglycqsylated human seminal plasma glycoprotein: 100 k~g/ml in 50 mM Tris-HCl (pH 7.5) with 0.1% SDS; A - - A - - A , further treatment with periodic acid (PI); D - - D - - D , pronase digestion following PI treatment.

This implies that certain periodate-sensitive moieties of deglycosylated HSP may be damaged. When the deglycosylated HSP antigens were treated with pronase or trypsin the antigenicity was markedly reduced.

Establishment of the hybridoma 3B2-F7 secreting sperm immobilizing monoclonal antibody which reacted to a peptide chain of human seminal plasma By using ELISA with coating of HSP glycoprotein deglycosylated by TFMS and followed by PI and enzymatic treatments, the supernatants from 11 highly positive hybridomas and a number of weakly reacting hybridomas were identified. The culture supernatants were tested in parallel by the sperm immobilization test (Isojima, 1976). The hybridoma secreting Ab which exhibited SI activity was named 3B2 (Table 1). After several clonings by limiting and sequential dilutions the clone named 3B2-F7 (IgM, K) showed SI activity as much as 256 SI50 units, while no SA activity was observed. Characterization of the epitope corresponding to Mab 3B2-F7 To clarify the characteristics of the antigen epitope corresponding to Mab 3B2-F7, the deglycosylated HSP antigen was treated with pronase, protease from bovine pancreas, subtilopeptidase A and proteas~ of Streptomyces griseus. The antigenicity corresponding to the,Mab was almost completely destroyed by these enzymes, and also partly by trypsin and V8 protease (Table 2). The antigenicity of deglycosylated HSP antigen was not diminished by the treatment with endoglycosidase D, glycopeptidase A and also the endoglycosidase N-glycanase (Peptide: N-glycosidase F) specific for asparagine-linked carbohydrate moieties. The antigen epitope corresponding to Mab 3B2-F7 proved to be in the approximately 36 kDa molecule as shown by Western blotting after SDSPAGE (Fig. 3). The 36 kDa component on SDS-PAGE containing antigen epitope to Mab 3B2-F7 was extracted from the SDS gel and reduced and alkylated. This was subjected to proteolysis with Staph. aureus V8 protease

TABLE 1 Total no. of wells growing hybridomas

No. of hybridomas secreting Mab positive in ELISA a

No. of cell clones exhibiting sperm immobilizing activity

587

11 (highly positive) 23 (moderately positive)

1b

aWells were coated with deglycosylated human seminal plasma glycoprotein (HPS-gp). bHybridoma 3B2.

TABLE 2 Sensitivity of epitope corresponding to Mab 3B2-F7 to enzymes and deglycosylating agents. Application of enzymes (A492) Antigenicity Before

After

Proteases Proteinase (from bovine pancreas) Proteinase (from Streptomyces griseus) Subtilopeptidase A Trypsin

1.82 1.93 1.87 1.94 1.86

0.127 0.087 0.082 0.183 0.328

Carbohydrate splitting enzymes Neuraminidase /3-Galactosidase /3-N-Acetylglucosaminidase /3-N-Acetylgalactosaminidase Mixed Glycosidases Endoglycosidase D Glycopeptidase A N-Glycanase (Peptide: N-Glycosidase F)

No No No No No No No No

change lchange change change change change change change

Deglycosylating agents Trifluoromethanesulfonic Acid (TFMS) Periodic Acid

Insensitive Insensitive

and trypsin. The resulting peptide fragments were applied to immunoaffinity chromatography bound Mab 3B2-F7. The bound peptides were eluted with acetic acid (pH 2.5) and their binding capabilities to Mab were examined by ELISA. The results showed that peptide fragments after enzyme digestion retained antigenicity (Table 3). These eluted antigenic peptide fragments were further fractionated by FPLC on Sepharose 4B and the sharp single band reactive to Mab 3B2-F7 was identified. Localization of the antigen epitope corresponding to Mab 3B2-F7 in reproductive and somatic tissues was performed by avidin-biotin peroxidase complex (ABC) staining (Fig. 4). Clear staining of the epithelium of human epididymis from caput to cauda portion and spermatozoa in the lumen of the epididymis was observed but the staining of testis was not. The Langerhan's islets of the pancreas, epithelial cells of the distal tubulus of the kidney were also stained. To avoid non-specific staining, the Mab was absorbed with human liver extract before use in the assay. Only slight staining of other somatic tissues was observed. The binding of 3B2-F7 to human spermatozoa was examined by ABC staining and immunofluorescence of paraffin-embedded sperm or motile non-fixed sperm. The staining reaction to sperm in the paraffin sections

10

1

2

- 94Kd

- 67Kd - 43Kd

- 30Kd

- 20.1Kd

- 14.4Kd

Fig. 3. Transblot to nitrocellulose membrane of human seminal plasma glycoprotein. 1, Amido Black staining; 2, immunochemical staining with Mab 3B2-F7.

was positive, while fresh non-fixed sperm were negative both by A B C staining and immunofluorescence. The biological effect of 3B2-F7 on human in vitro fertilization (IVF) was examined. Table 4 shows that sperm binding and penetration of zona pellucida were strikingly blocked when 3B2-F7 was added to the IVF system. TABLE 3 Antigenicity of peptide fragments after digestion with proteolytic enzyme. Antigen cleaved with

Enzyme digestion Antigenicitya

Staph. aureus V8 Protease

Trypsin aElution from Mab bound column. bA492 estimated in ELISA.

Before

After

2.19 b 1.97

2.45 2.36

11

Fig. 4a,b.

Discussion The Mab 3B2-F7 reacting to an apparent peptide determinant of human seminal plasma glycoprotein (36 kDa) was selected owing to its reactivity to TFMS-deglycosylated HSP antigen which was resistant to periodate and

12

tl

Fig. 4. Immunohistochemical (ABC) staining of various tissues. (a) Human epididymis (×200); (b) Human kidney (x 100); (c) Human pancreas (x 40); (d) Human testis (x 100).

13 TABLE 4 Effects of Mab 3B2-F7 on binding and penetration of human sperm to zona pellucida (Z.P.) No. of eggs examined Supernatant of P3U1 Mab 3B2-F7

9 12

No. of sperm bound on Z.P.

No. of sperm penetrated into Z.P.

72.1 -- 13.0a (53-96) 20.6 ± 16.8 (2-65)

3.2 -- 1.2 (1-4) 0.6 -- 0.8 (0-2)

aMean -+ S.D. (ranges). In vitro maturated and denuded human follicular oocytes were transferred to driplets of pre-incubated human sperm suspension (1 x 106/ml) in the medium containing 50% culture supernatant of mouse myeloma (P3U1) or hybridoma (3B2-F7) and incubated 15-18 h at 37°C in 5% CO2 incubator. After incubation, the number of spematozoa bound or penetrated into zona pellucida was counted under a phase-contrast microscope.

exo-and endoglycosidases. HSP proteins excreted from the epithelium of the epididymis may be altered by variable degrees of glycosylation during transfer through the epididymal duct. Reports on the deglycosylating agent TFMS provide data on the efficient cleavage of 0-glycosyl linkage while Nglycosyl bonds of asparagine-linked glucosamine and galactosamine seem to remain intact (Edge et al., 1981; Woodward et al., 1987). From our results, our target antigen molecule seems not to be N-linked carbohydrate, as N-glycanase cleavage of TFMS-deglycosylated and periodate-treated target antigen resulted in no detectable loss of antigenicity. The TFMStreated HSP antigens immobilized on the plate in ELISA may provide several core proteins bearing oligosaccharide chains of variable length and structure. Therefore, if Mabs against such oligosaccharide moieties are raised, the ELISA may show positive reactions in screening. ELISA is a well established and favoured technique to detect various antigens by using corresponding antibodies but has certain limitations for hydrophobic proteins for coating on the plastic plate. But when detergent solubilization was used for such hydrophobic proteins, efficient coating could be achieved by covalent coupling to plastics (Newman et al., 1981). In such a case the detergent must not interfere with protein attachment (Gardas et al., 1988; McGabe et al., 1988). Immobilization of HSP deglycosylated target antigens was achieved in the presence of a detergent with high CMC value and this contributed to the detection of hybridomas against a peptide chain of HSP antigens. Hybridoma 3B2-F7 which secreted SI-Mab was selected, thus Mab 3B2-F7 seems to identify a peptide epitope of HSP-gP on human sperm which is relevant to sperm immobilization. Staining patterns of human sperm with this Mab depend-on the exposure of the peptide epitope on the sperm membrane. However, the occurrence of sperm immobilization by 3B2-F7 in the presence of complement suggests

14

the existence of a peptide epitope which is exposed on some regions of the sperm membrane, though the majority of the surface may be covered with carbohydrate components. This possibility could be explained by the fact (unpublished results) that sperm from some men were immobilized by 3B2F7 whilst the sperm from others were not. This implies that some ejaculated sperm were not covered overall with carbohydrate substances and that there are some exposed areas of peptide epitopes on the surface, though the other type of ejaculated sperm might be covered with carbohydrate components over the entire surface. These results strongly suggest that peptide epitopes of HSP glycoprotein coated on the sperm surface may be relevant to sperm immobilization. The interaction of secreted products from the male accessory glands with sperm might have a role in sperm maturation during transit through the epididymis and seminal tract (Horan and Bedford, 1972; Kohane et al., 1980; Moore, 1980; Feuchter et al., 1981; Peterson et al., 1984; Ellis et al., 1985.). Our unpublished results indicated that the antigen epitope corresponding to 3B2-F7 was retained after treatment of ejaculated sperm with N-glycanase and periodate. The antigen epitope corresponding to 3B2-F7 was sensitive to multiple proteases but resistant to TFMS, periodate and splitting by carbohydrate-specific enzymes. The target antigen epitope for 3B2-F7 seems to be a continuous peptide epitope and not to be of a polypeptide conformation as the antigenicity was retained by digestion with some proteolytic enzymes after SDS-PAGE. When the target HSP antigens are immobilized under denaturing conditions in ELISA assay the peptide epitopes are likely to be more exposed on the surface of wells. However, our results show that the target antigen epitope seems to be a peptide sequence in 36 kDa HSP glycoprotein which may be secreted from epididymal epithelium. Analysis of the amino acid sequence of the target antigen is now underway. In addition to sperm immobilization, Mab 3B2-F7 seems to have the biological activity of inhibiting sperm binding to zona pellucida in the human IVF system. Therefore, the corresponding antigen may have some role in sperm interaction with zona pellucida. Mab 3B2-F7 may provide a probe for the screening of the epididymal c D N A library and isolation of the corresponding c D N A clone. The existence of the target antigen epitope in tissues other than epididymal epithelium, especially in the islets of Langerhan's cells of the pancreas and the epithelium of the distal urinary tubules of the kidney, is a problem. However, the presence of the epitope was observed after fixation, thus there is no evidence as to whether or not the epitope is exposed in these tissues in vivo. We have strong supporting evidence that the high titer of circulating SIantibody against HSP in common with red blood cells and kidney tubules does not have any harmful clinical effects over a long observation period,

15

though localization of this antigen epitope was demonstrated in vitro after fixation of these tissues. From these results, the possible side effects of Mab 3B2-F7 in vivo is still questionable and further study is required of the dynamic exposure of the antigen molecules in vivo. References Edge A., Faltynek C., Hof.L., Reichert L. and Weber P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem. 118, 131-137. Ellis D.H., Hartman T.D. and Moore H.D.(1985) Maturation and function of the hamster spermatozoon probed with monoclonal antibodies. J. Reprod. Immun. F., 299-314. Fairbanks G., Steck T. and Wallach D. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochem. 10, 2606--2617. Feuchter F.A., Vernon R.B. and Eddy E.M. (1981) Analysis of the sperm surface with monoclonal antibodies: Topographically restricted antigens appearing in the.epididymis. Biol. Reprod. 24, 1099-1110. Franklin R. and Dukes C.D. (1964) Antispermatozoal antibody and unexplained infertility. Am. J. Obst. Gynec. 89, 6--9. Gardas A.and Lewartowska A. (1988) Coating of proteins to polystyrene ELISA plates in the presence of detergents. J. Immun. Meth. 106, 251-256. Horan A.H. and Bedford.T.M.(1972) Development of the fertilizing ability of spermatozoa in the epididymis of the Syrian hamster. J. Reprod. Fert. 30, 417--423. Isojima S. and Koyama K.(1976) Quantitative estimation of immobilizing antibody in the sera of women with sterility of unknown etiology; the 50% sperm immobilization unit (SI50). Recent Advances in Human Reproduction pp.10-15. (A. Campos da Paz, V.A. Drill, H. Hayashi, W. Rodoiques and A.V. Schally eds.) Excerpta Medica, Elsevier, Amsterdam. Isojima S. and Koyama K.(1978) Microtechnique of sperm immobilization test. In:Immunology of Reproduction (Bratanov K. et al., eds.), pp 215-219. Bulgarian Academy of Sciences, Sofia. Isojima S., Koyama K. and Fujiwara N.(1982) Purification of human seminal plasma No.7 antigen by immunoaffinity chromatography on bound monoclonal antibody. Clin. Exp. Immun. 49, 449-456. Isojima S., Kameda K., Tsuji Y., Shigeta M., Ikeda Y. and Koyama K.(1987) Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma. J. Reprod. Immunol. 10, 67-78. Kohane A.C., Canuo M.S., Cimeito L., Carberi T.C. and Blaquiz J.A. (1980) Distribution and site of production of specific proteins in the rat epididymis. Biol. Reprod. 23, 181-187. Koyama K., Hasegawa A., Tsuji Y. and Isojima S. (1985) Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae. J. Reprod. Immun. 7, 187-198. Kyurkchiev S.D., Shigeta M., Koyama K. and Isojima S. (1986) A human-mouse hybridoma producing monoclonal antibody against human sperm-coating antigen. Immunology 57, 489-492. Laemmli U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, Lond., 227, 680~585. McCabe J., Fletcher S. and Jones M. (1988) The effect of detergents on the ELISA of blood group substances. J. Immun. Meth. 108, 129-135. Moore H.D. (1980) Localization of specific glycoproteins secreted by the rabbit and hamster epididymis. Biol. Reprod 2 2, 705-718. Newman P., Kahn R. and Hines A.(1981) Detection and characterization of monoclonal antibodies. J. Cell Biol. 90, 249-257. Peterson R., Russell L. and Hunt W. (1984) Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro. J. Exp. Zool 231, 137-147. Shigeta M., Watanabe T., Maruyama S., Koyama K. and Isojima S. (1980) Sperm-immobilizing monoclonal antibody to human seminal plasma antigens. Clin. Exp. Immunol., 42, 458--462.

16 Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350--4354. Tsuji Y., Clausen H., Nudelman E., Kaizu T., Hakomori S.-I. and Isojima S. (1988) Human sperm carbohydrate antigens defined by an anti-sperm human monoclonal antibody derived from an infertile woman bearing anti-sperm antibodies in her serum. J.Exp. Med,, 168, 343-356. Woodward H., Ringter N., Selvakumar R., Simet I., Bhavanandan V. and Davidson E.(1987) Deglycosylation studies on tracheal mucin glycoproteins. Biochem. 26, 5315-5321.