Monoclonal antibodies to the carboxy-terminal Ea sequence of pro-insulin-like growth factor-IA (proIGF-IA) recognize proIGF-IA secreted by IM9 B-lymphocytes.

Insulin-like growth factor-I (IGF-I) circulates in human serum as a 7 kDa peptide but analysis of IGF-I cDNAs predicts two pro-hormone precursors (proIGF-IA and proIGF-IB) with distinct C-terminal E domains. The function of these precursors, and the E peptides generated on cleavage to mature IGF-I, is unknown, largely because of a lack of tools for distinguishing precursors from constituent peptides. We used a synthetic Ea peptide to develop monoclonal antibodies (MAbs) which can recognize the carboxy-terminal sequence of proIGF-IA. These were characterized using proIGF-IA generated by transfected HEK293 cells. The anti-proIGF-IA MAbs immunoprecipitated two peptides (19--21 and 14 kDa) which were also recognized by MAbs to mature IGF-I. The proIGF-IA MAbs could also detect peptides of 9 and 4 kDa predicted to be Ea peptides. Treatment with N -glycosidase proved the 19--21 kDa and 9 kDa bands to be glycosylated proIGF-IA and Ea peptide respectively. Using these antibodies, we have identified proIGF-IA secreted from the IM9 B-lymphocyte cell line. This work paves the way for studies on proIGF-IA and Ea peptide regulation and function. Copyright 2001 Harcourt Publishers Ltd.