Analytical performance of a sandwich enzyme immunoassay for preβ1-HDL in stabilized plasma

Abstract

We have established an immunoassay for pre ␤ 1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because pre ␤ 1-HDL is unstable during storage, fresh plasma must be used for pre ␤ 1-HDL measurements. In this study, we describe a method of stabilizing pre ␤ 1-HDL, and evaluate the analytical performance of the immunoassay for pre ␤ 1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Pre ␤ 1-HDL concentration was measured by immunoassay. In nonpretreated samples, pre ␤ 1-HDL decreased significantly from the baseline after 6 h at room temperature. Although pre ␤ 1-HDL was more stable at 0 ؇ C than at room temperature, it increased from 30.2 ؎ 8.5 (SE) to 56.5 ؎ 5.5 mg/l apolipoprotein A-I (apoA-I) ( P Ͻ 0.001) in hyperlipidemics, and from 18.4 ؎ 1.2 to 37.9 ؎ 3.3 mg/l apoA-I ( P Ͻ 0.001) in normolipidemics after 5-day storage. After 30-day storage at ؊ 80 ؇ C, pre ␤ 1-HDL increased from 29.0 ؎ 4.0 to 38.0 ؎ 5.7 mg/l apoA-I ( P Ͻ 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, pre ␤ 1-HDL concentration did not change significantly under any of the above conditions. Moreover, pre ␤ 1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n ‫؍‬ 24, r ‫؍‬ 0.833, P Ͻ 0.05).An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of pre ␤ 1-HDL.

Structure of HDL: Particle Subclasses and Molecular Components

“…Agarose gels can be stained with Coomassie blue or with anti-apolipoprotein A-I (apoA-I) antibodies, and the relative protein content of the two HDL subclasses can be determined (Favari et al 2004). The plasma preβ-HDL concentration can be also quantified using a sandwich enzyme immunoassay (Miida et al 2003). The assay utilises a monoclonal antibody which specifically recognises apoA-I bound to preβ-HDL.…”

Physical Fitness and Reverse Cholesterol Transport

“…These strict conditions were essential because keeping plasma at ϩ4°C for Ͼ3 hours, as well as prolonged storage at Ϫ20°C, slow freezing, or repeated freezing and thawing of plasma lead to elevation of pre␤ 1 -HDL levels, probably because of decay of mature HDL particles. 10 Lipids, Lipoproteins, Cholesteryl Ester Transfer Protein, and Lecithin:Cholesterol Acyltransferase Plasma total cholesterol (TC), triglyceride (TG), LDL cholesterol, apolipoprotein B (apoB), HDL cholesterol, and apolipoprotein A-I (apoA-I) were measured using spectrophotometric techniques with a Cobas-FARA centrifugal analyzer (Roche Diagnostic Systems). Pre␤ 1 -HDL concentration was measured by enzyme-linked immunosorbent assay (ELISA) 11 (Daiichi Pure Chemicals, Tokyo, Japan).…”

“…Changes in concentration of pre␤ 1 -HDL determined by ELISA correlated with changes determined by 2-dimensional nondenaturing electrophoresis. 10 Cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT) concentrations were measured by ELISA (Daiichi). CETP activity was measured by fluorometric assay (Roar Biomedical, New York, NY).…”

An apolipoprotein A-I mimetic dose-dependently increases the formation of preβ1 HDL in human plasma

“…This HDL subclass exhibits preb mobility in nondenaturing gel electrophoresis and is postulated to consist of two to three apolipoprotein A-I (apoA-I) molecules with some phospholipids and a small amount of unesterified cholesterol (9). As preb 1 HDL takes on unesterified cholesterol from cell membranes, it exhibits unique epitopes not exposed in spheroidal a-HDL (10)(11)(12).…”