Journal of Lipid Research, Vol 29, 85-93, Copyright © 1988 by Lipid Research, Inc.
Apolipoprotein A-I assayed in human serum by isotope dilution as a potential standard for immunoassay
PK Weech, D Jewer and YL Marcel
Clinical Research Institute of Montreal, Laboratory of Lipoprotein Metabolism, Quebec, Canada.
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.
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