Abstract
Previous attempts to prepare monoclonal antibodies (MAbs) against S-antigen, a photoreceptor cell protein involved in the visual process and a potent autoantigen for the induction of experimental autoimmune uveitis (EAU), have yielded MAbs which define only carboxyl terminal epitopes. In this study we devised alternate strategies to prepare five MAbs directed to other regions of the molecule. MAbC10C10 and MAbH11-A2 were prepared against synthetic peptides known to be uveitopathogenic and they were selected for more detailed studies. MAbC10C10 was generated against synthetic peptide BSA281-302 which contains a predictive consensus sequence for defined T cell epitopes (GIALD) as well as a consensus sequence for GTP-binding proteins. One human adenosine deaminase synthetic peptide containing an extensive amino acid sequence homology to BSA281-302 was a potent inhibitor of MAbC10C10 binding in a competitive inhibition radioimmunoassay. MAbH11-A2 was generated against peptide BSA303-332 which also contains a uveitopathogenic site. The binding site of MAbH11-A2 was determined to be within amino acid positions 305 to 314 (NLASSTIIKE) in S-antigen. This binding site corresponded closely to the binding site of an affinity-purified rat polyclonal antibody raised to human S-antigen. MAb5C6.47 was isolated from a mouse hyperimmunized with bovine S-antigen and was specific for a highly conserved sequence near the amino terminus, amino acid residues 42 to 48 (DGVVLVD). Both MAbC10C10 and MAb5C.47 were useful in screening gtll cDNA libraries expressing S-antigen polypeptides as fusion proteins. Our results demonstrate the feasibility of producing site-specific MAbs potentially useful in the study of T cell-mediated immune mechanisms in EAU as well as in the phototransduction of vision.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 343-355 |
| Number of pages | 13 |
| Journal | Current Eye Research |
| Volume | 9 |
| Issue number | 4 |
| DOIs | |
| State | Published - 1990 |
Bibliographical note
Funding Information:Leonard Foster, Joyce D. Fuhrman and Joy Whitman are greatly appreciated. Supported in part by the Retina Service of Wills Eye Hospital, the Pennsylvania Lions Sight Conservation and Eye Research Foundation, the Fannie Rippel Foundation, NIH grants EY05095, EY07737, EY05417 and BRSG5510, the Crippled Children's Vitreo-Retinal Research Foundation (David Meyer, Director), the Elizabeth C. King Trust (TS) and the Harry B. Wright Trust (TS). Dr. Gregerson is a Research to Prevent Blindness Senior Scientific Investigator. Dr. Vrabec is the Henry and Corinne Bower Retina Research Scholar. Dr. Donoso is the Thomas D. Duane Research Professor of Ophthalmology, Jefferson Medical College, Thomas Jefferson University and the recipient of Manpower Award from Research to Prevent Blindness. MAbC5.47 requests to Dr. Smith and MAbClOClO antibody requests to Dr. Donoso.
