Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay

• Main Authors: G Schonfeld, M S Frick, A P Bailey
• Format: Article
• Language: English
• Published: Elsevier 1976-01-01
• Series: Journal of Lipid Research
• Subjects: rat high density lipoprotein; apolipoprotein A-I; radioimmunoassay
• Online Access: http://www.sciencedirect.com/science/article/pii/S0022227520370127

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and > 99% of bound 125I-apoA-I was displaced by “cold” apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.Rat intact HDL displaced 125I-apoA-I in parallel with “cold” apoA-I; however, only about 10% of the apoA-I in intact HDL reacted in the assay. Removal of the lipid from HDL by extraction with ether-ethanol solutions increased the reactivity of apoA-I to levels expected from studies of the apeA-I content of HDL by column chromatography and SDS disc gel electrophoresis. Plasmas from Sprague-Dawley male and female rats also displaced counts in parallel with apoA-I. Apparent plasma levels of apoA-I ranged from 2 to 7 mg/dl in unextracted plasma. When assay incubations were carried out for two hours a t 37°C at theb eginning of the assay and again right after the addition of the second antibody, similar low results were obtained. Lipid extraction increased apparent levels about 9-fold to 39 ± 8 mg/dl for Sprague-Dawley males (n = 14) and 47 ± 6 mg/dl for females (n = 5). ApoA-I was not lost during extraction. Heating of plasmas to 52°C before assay yielded levels of 26 ± 4 mg/dl. Thus, extraction of plasmas appears to be necessary to obtain accuralteev els of apoA-I in this assay system.