A rapid method of epitope mapping. Application to the study of immunogenic domains and to the characterization of various forms of rabbit progesterone receptor.
A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C-terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C-terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action. This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1-60, 101-110, 295-325 and 370-396. There were no antibodies directed against the DNA-binding or the steroid-binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross-reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1-60). The 79-kDa form ('subunit A') of the receptor was shown to lack the two more N-terminally localized immunogenic domains (amino acids 1-60 and 101-110). The 65-kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N-terminal part of the protein.
Lorenzo
F
F
Hormones et Reproduction (Institute National de la Santé et de la Recherche Médicale Unité 135), Hôpital de Bicêtre, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre, France.
Jolivet
A
A
Loosfelt
H
H
Thu vu Hai
M
M
Brailly
S
S
Perrot-Applanat
M
M
Milgrom
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D000939:Epitopes / Q000032:analysis*; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D007120:Immunochemistry; D007150:Immunohistochemistry; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D011980:Receptors, Progesterone / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011980:Receptors, Progesterone; D004247:DNA; D004262:DNA Restriction Enzymes