Mapping of the Epitope Recognized by a Monoclonal Antibody Specific for RPE65 and the Retinal Pigment Epithelium

Abstract

Abstract: : Purpose: Mutations in RPE65 are a cause of early-onset severe retinal dystrophy including Leber congenital amaurosis. We developed a monoclonal antibody against RPE65 that has been widely distributed and used in various applications. We now report on epitope mapping and RPE specificity of this antibody. Methods: Bovine RPE microsomal membranes were prepared and used to generate a panel of monoclonal antibodies by standard methods. RPE65 was partially purified by SDS gel electrophoresis, and tryptic digests were analyzed by western transfer and MALDI-TOF protein mass spectrometry. Yeast strains were made by transfection with pHybLex/Zeo expression contructs containing ~300 bp fragments of RPE65 cDNA sequence. Synthetic peptides corresponding to RPE65 sequences were generated and assayed by competition ELISA and immunoprecipitation. Immunohistochemistry was performed on eye cryosections. Results: A hybridoma (8B11.37) secreting an antibody with high specificity for RPE65 was isolated and IgG purified from ascites fluid. A ~17 kD antigenic peptide was identified by analysis of RPE65 tryptic digests. An overlapping ~12 kD antigenic peptide was identified by analysis of yeast strains expressing RPE65 partial sequence. A 16 aa peptide corresponding to sequence within the ~12 kD region competed antibody binding with µM affinity in ELISA and immunoprecipitation experiments. RPE65 reactivity was detected only in the RPE cell layer of eye sections from bovine, human, mouse, and Xenopus laevis, as well as from Nrl homozygous null mice whose retinas contain increased numbers of S cone-like cells and lack rod cells. Conclusions: The identification of an RPE65 antigenic epitope is a first step toward developing a structural understanding of this important disease gene product, as well as an affinity method for protein purification. The RPE-restricted reactivity of the 8B11.37 monoclonal antibody in eye sections from a number of species does not support the proposed involvement of RPE65 in the cone cell visual cycle.

Keywords: retinal degenerations: hereditary • retinal pigment epithelium • proteins encoded by disease genes