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.
Donoso
L A
LA
Retina Service, Wills Eye Hospital, Philadelphia, PA.
Rodrigues
M
M
Vrabec
T R
TR
Sery
T W
TW
Fong
S L
SL
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