Failure of acid-ethanol treatment to prevent interference by binding proteins in radioligand assays for the insulin-like growth factors.
Acid-ethanol precipitation and gel filtration at acidic pH have been widely used to extract circulating binding proteins for insulin-like growth factor (IGF-I and IGF-II) from plasma or serum samples before radioligand assay for the respective IGFs. Gel filtration on Sephadex G-50 at neutral pH of neutralized acid-ethanol extracts of fetal and adult ovine plasma which had been incubated with 125I-labelled IGF-I or 125I-labelled IGF-II revealed that significant amounts of the IGF-binding protein activity survived the acid-ethanol extraction procedure. Radioimmunoassay for IGF-I in acid-ethanol extracts of plasma samples from fetal, neonatal and adult sheep yielded results which depended upon the method used for separation of the antibody-bound IGF-I tracer from the free IGF-I tracer. Acid gel filtration of ovine fetal and adult plasma was found to remove completely the IGF-binding protein activity. Radioimmunoassay for IGF-I in samples of fetal, neonatal and adult sheep plasma that had undergone acid gel chromatography yielded consistent results for both methods that were used to separate antibody-bound IGF-I tracer from the free tracer. Radioreceptor assays for IGF-II were similarly highly perturbed by the presence of binding protein in acid-ethanol extracts of ovine fetal and adult plasma. We conclude that acid-ethanol extraction can not be used reliably for the removal of IGF-binding proteins, and that only acid gel filtration is a completely safe and valid method.
Mesiano
S
S
Department of Physiology, Monash University, Clayton, Victoria, Australia.
Young
I R
IR
Browne
C A
CA
Thorburn
G D
GD
D000143:Acids; D000818:Animals; D002352:Carrier Proteins / Q000097:blood*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D000431:Ethanol; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018969:Insulin-Like Growth Factor Binding Proteins; D008297:Male; D011869:Radioligand Assay / Q000379:methods*; D051381:Rats; D012756:Sheep; D013002:Somatomedins / Q000097:blood*
D000143:Acids; D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D013002:Somatomedins; D000431:Ethanol