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a Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
b Laboratory of Developmental Biology and Reproduction, Institute of Advance Medical Sciences, Nishinomiya, Hyogo 663, Japan
c Department of Obstetrics and Gynaecology, Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan
To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136–147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136–147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136–147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.
1 This work was supported by a grant from the Department of Biotechnology, Government of India. Support for a part of this work (CSA-98–219/CSA-99–262) was also provided by the Contraceptive Research and Development (CONRAD) Program, Eastern Virginia Medical School, Norfolk, VA. The views expressed by the authors do not necessarily reflect the views of the funding agencies.
2 Correspondence. FAX: 91 11 6162125; skgupta{at}nii.res.in
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