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Vaccination has stood the test of time-over the decades for control of variety of viral, bacterial and other infectious diseases. It was therefore deemed logical to devise strategies to vaccinate against conception using a panel of antigens expressed along the entire pituitary, hypothalamic and gonadal axis. Immunocontraception represents an attractive mode of fertility regulation because of its potential for safety, reversibility, efficacy and low-cost wide usage, and global acceptability. Rapid progress in understanding the immune system and availability of an impressive assay of tools for the production of synthetic and recombinant proteins provides a strong foundation for the development of a molecular or subunit vaccine.
Sperm antigens are being investigated for their potential use in contraceptive vaccines. A few sperm specific antigens have been purified, characterized and some of them are relevant to fertility. For vaccine induced antisperm antibodies to inhibit the function of viable sperm, target antigens must be expressed on the sperm surface.
The study was conducted in an effort to identify the most relevant antigens of human sperm membrane with a potential role in fertility. Plasma membrane was isolated from human spermatozoa. When subjected to one dimensional SDS-PAGE, the membrane fraction resolved into discrete polypeptides with molecular weight range from 15 kDa to Immunogenicity 120 kDa. Rabbits were immunized with the membrane preparation to cheeky ELISA revealed high antibody litres in rabbits as was reconfirmed by positive fluorescence on human spermatozoa.
The antibody raised against whole membrane was checked for agglutinating and immobilizing activity using human spermatozoa. It was found that the antisera had both agglutinating as well as immobilizing activity and more importantly had the capacity to inhibit sperm-egg interaction in vitro.
The most dominant antigens of human sperm membrane were identified using infertile as well as hyperimmune rabbit sera in western blot assays. Two immunodominant antigen belonging to 26 kDa and 46 kDa family of proteins were identified. The proteins were electro eluted from the polyacrylamide gels and were used to raise monospecific antibodies in rabbits. The immune sera were characterized using ELISA, immunofluorescence which showed the presence of antibodies against 26 kDa and 46 kDa protein. Both the sera had potent effects on agglutination and immobilization of human spermatozoa. Gamma globulins were isolated from the immune sera raised against 26 kDa and 46 kDa proteins using DEAE-cellulose. The separated IgG were tagged to Sepharose 4B-CI column to affinity purify the 26 kDa and 46 kDa proteins from whole sperm membrane. The affinity purified antigens showed a single band in the SDS-PAGE. The antibodies to 26 kDa inhibited hamster oocyte penetration by human spermatozoa, while antibodies to 46 kDa although active in agglutination and immobilization assays failed to inhibit sperm oocyte penetration assay.