Insulin-Like Growth Factor Carrier Proteins in Neonatal and Adult Rat Serum Are Immunologically Different: Demonstration Using a New Radioimmunoassay for the Carrier Protein from BRL-3A Rat Liver Cells

Abstract

A carrier protein for insulin-like growth factors (IGFs) has been purified from serum-free medium conditioned by the Buffalo rat liver (BRL)-3A cell line and used to immunize rabbits. Purified carrier protein was 125I labeled and affinity purified on IGF-Sepharose. The major labeled protein had a mol wt of about 33,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate for the IGF carrier protein subunit) and gave a single predominant peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gel electrophoresis that was immunoprecipitated by immune serum and comigrated with unlabeled proteins that bind [125I] IGF. A RIA was developed using affinity purified [125I]carrier protein and immune serum. Tracer binding was inhibited only by preparations containing IGF carrier proteins, but not by unrelated proteins or by the IGFs themselves. Carrier proteins from BRL-3A cells gave equivalent strong reactivity either after dissociation of endogenous IGF or as an IGF-carrier protein complex. The antiserum effectively recognized the approximately 40,000 mol wt (Mr approximately 40,000) carrier protein from neonatal rat serum, both as a native complex and after acid stripping. It did not effectively recognize the Mr approximately 150,000 carrier protein from adult rat serum either as endogenous complex or after acid stripping. These results suggest that the Mr approximately 40,000 carrier protein of neonatal rat serum and the Mr approximately 40,000 binding subunit of the Mr approximately 150,000 carrier protein in adult rat serum are immunologically distinct. These antisera to the BRL-3A carrier protein should be useful tools with which to dissect the relationships between different carrier protein species and to study the regulation of IGF carrier protein gene expression.