Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity.
Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of Mr 150,000 and the other of Mr 40,000-45,000. The majority of the immunoreactive IGF-I was associated with the Mr 150,000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150,000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose-response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment.
Lee
C Y
CY
Department of Animal Science, Clemson University, South Carolina 29634-0361.
Henricks
D M
DM
D000143:Acids; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000378:metabolism; D008297:Male; D011863:Radioimmunoassay
D000143:Acids; D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I