IRBP: preparation and characterization of site-specific monoclonal antibodies.
Interstitial retinoid binding protein (IRBP) is a 136,000 molecular weight photoreceptor cell protein which is a highly pathogenic autoantigen for the induction of experimental autoimmune uveitis (EAU). In this study we produced a series of monoclonal antibodies (MAbs) which define different epitopes in the native molecule. These MAbs were further subdivided into three distinct groups based on a radioimmunoassay, and by ELISA assay using native IRBP and synthetic peptides corresponding to its entire amino acid sequence. Group I MAbs (MAbD7-B1 and MAbC6-B4) bound to native IRBP but not to any synthetic peptides, suggesting that their antigenic epitopes are strictly conformation dependent. Group II MAbs (MAbC7-D3 and MAbG8-H4) bound weakly to multiple peptides which shared amino acid sequence similarity located within each of four homology domains indicating that these epitopes are also conformation dependent. In group III (MAbH3-B5, MAbH7-A5, and MAbB6-D12) MAb binding was localized to a specific peptide. The MAbH3-B5 binding site was further refined to amino acid positions 361 to 367 in the native molecule. MAbH3-B5 was also useful in localizing IRBP in the mouse retina by immunohistochemical techniques. The application of these MAbs in the study of EAU and interphotoreceptor transport mechanisms is discussed.
Retina Service, Wills Eye Hospital, Philadelphia, PA.
L A
LA
Donoso
M
M
Rodrigues
T R
TR
Vrabec
T W
TW
Sery
S L
SL
Fong
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000276:immunology; D001667:Binding, Competitive; D000939:Epitopes / Q000276:immunology; D005136:Eye Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D012177:Retinol-Binding Proteins / Q000276:immunology*; D000099214:Retinol-Binding Proteins, Interstitial
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005136:Eye Proteins; D010455:Peptides; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial