Apolipoprotein A-I assayed in human serum by isotope dilution as a potential standard for immunoassay.
We measured the amount of apoA-I in serum by isotope dilution, finding 1.33 mg/ml (standard deviation 0.177) in six normolipidemic, healthy subjects. We developed this method by adapting published techniques to purify apoA-I from 3 ml of serum in two steps: density gradient ultracentrifugation and high performance liquid chromatography gel filtration. The 125I-labeled apoA-I tracer was first screened, by incubation with serum, to select labeled apoA-I which retained the ability to exchange with native apoA-I and bind to HDL. A known amount of 125I-labeled apoA-I-labeled HDL was added to unknown serum samples; apoA-I was reisolated from the serum and its specific radioactivity was used to calculate the dilution of the added, labeled apoA-I by the unlabeled apoA-I in the unknown serum. By not relying on immunochemical techniques, the isotope dilution assay provided results that are independent of the expression of individual apoA-I antigenic sites. Therefore, sera that have been assayed by isotope dilution can serve as standards to evaluate the accuracy of immunoassays for serum apoA-I and provide primary standards for such immunoassays.
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood; D008075:Lipoproteins, HDL / Q000302:isolation & purification; D011863:Radioimmunoassay / Q000379:methods*; D011865:Radioisotope Dilution Technique
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL