Cholesterol determination in serum after fractionation of lipoproteins by immunoprecipitation.

Abstract

We investigated the immunoprecipitation of apolipoprotein B-binding lipoproteins, or of apo A-I- and apo C-binding lipoproteins, by delipidated antiserum for measuring cholesterol in the nonprecipitated lipoprotein fractions. After immunoprecipitation of serum with delipidated anti-apo B, we determined by immunoelectrophoresis that no beta- or pre-beta lipoproteins were present, whereas alpha-lipoproteins remained in the supernate. Conversely, after immunoprecipitation with an antiserum against apo A-I + apo C, only lipoproteins with beta-mobility were detected and no apo B from beta-lipoproteins was in the precipitate. The concentration of cholesterol in the supernate after immunoprecipitation with anti-apo B correlated highly (r = 0.93, n = 118) with cholesterol measured after precipitation with phosphotungstic acid/MgCl2. The cholesterol concentration after immunoprecipitation with anti-apo AI and anti-apo C correlated similarly well (r = 0.94, n = 145) with the LDL-cholesterol calculated by Friedewald's formula in serum specimens reflecting moderate hyperlipoproteinemia.

Clinical chemistry

“…Problems involved are lack of standardized procedures, lack of reliable quality control materials, changing immunoreactivity due to self-association and aggregation of the particles, varying content of lipid which may mask epitopes, and different affinities of the antisera. Assays and assay modifications have been recently described for apoA-I (350)(351)(352)(353)(354), apoB (352,353,(355)(356)(357)(358), Lp(a) (359), apoC (353), apoC-III (360,361). Reference ranges are not yet well defined, especially those of apolipoproteins (362,363).…”

Amperometric Determination of Serum Cholesterol with Pencil Graphite Rod

“…To check the reproducibility and reliability of the methods, the cholesterol content of the sample in one run (Within batch) and after storage at -20˚C for one week (Between batch) were determined. The results showed that the cholesterol value of these determination agreed with each other and within batch and between batch coefficient of variation (CV) were 1.59% & 4.15 % (Table 2), which is quite close to earlier reports such as colorimetric, electrochemical method [26] employing alkyl amine glass bound enzyme (1.6% for intrabatch and 3.2% for interbatch), amperometric method using silica gel bound enzyme (< 1.5% for all samples) [7] and flow injection method employing controlled pore glass bound cholesterol esterase and cholesterol oxidase (within day < 1.0 and between day < 2.5%) [27], measuring cholesterol after precipitation with phosphotugstic acid/MgCl 2 (within day 5.0 % and between day 8.2%) [28] and amperometric detection of cholesterol (within day 2%-between day 4%) [9]. The low coefficient of variation values indicated the accuracy, reproducibility and reliability of the method.…”