Abstract
A single radial immunodiffusion (SRID) method was applied to determine the concentrations of apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II).
ApoA-I and apoA-II were purified and used to prepare monspecific antisera by immunizing rabbits. An aliquot of high density lipoprotein (HDL) was delipidated with an equal volume of tetramethylurea (TMU), and then diluted with 8M urea in 10mM Tris-HCl buffer, pH 8.0. Agarose SRID plates were prepared by using the specific antisera. 4μl of each sample were added in duplicate wells on the plates which were placed in a humid chamber for 48hr. The diameter of the ring-shaped immunoprecipitates was measured in 0.1mm units.
The concentrations of apoA-I and apoA-II measured after delipidation with TMU and subsequent dissolution in 8M urea were 98 and 94%, respectively, of the concentrations yielded by delipidation with ether/ethanol and subsequent dissolution in 8M urea. The precipitate rings attained their final size within 24hr at room temperature. Intrassay coefficients of variation [(SD/mean)×100] within one plate for apoA-I and apoA-II analyses were 7.2 and 2.5%, and those between plates were 2.5 and 8.5%, respectively.
Results presented in this paper show that the SRID method is acurate, technically simple, and applicable to measurement of these apolipoproteins (apoA-I and apoA-II) in human plasma HDL.
