AnalyticalSimple and practical sandwich-type enzyme immunoassay for human oxidatively modified low density lipoprotein using antioxidized phosphatidylcholine monoclonal antibody and antihuman apolipoprotein-B antibody
Introduction
Oxidatively modified low density lipoprotein (Ox-LDL) is thought to play important roles in the early development of atherosclerosis through recruitment of monocyte-macrophages in vessel walls and accumulation of esterified cholesterol in the cells 1, 2. Ox-LDL has the ability to bind scavenger receptors, which can lead to unregulated accumulation of cholesterol in macrophages and the formation of foam cells. Thus, formation of lipid-laden foam cells from macrophages has been demonstrated by incubating Ox-LDL with macrophages in vitro (3), while incubation with native low density lipoprotein (LDL) did not result in accumulation of lipid droplets. Epitopes characteristic of Ox-LDL are detectable in atherosclerotic lesions (4). LDL extracted from atherosclerotic lesions has been shown to share many of the characteristics of copper-induced Ox-LDL (5). In addition, Ox-LDL is believed to be directly associated with atherosclerosis. Ox-LDL is cytotoxic (6) and induces various cellular responses such as vasocontraction (7), cellular proliferation (8), and expression of adhesion molecules (9).
It was generally assumed that little Ox-LDL is present in plasma and that Ox-LDL in atherosclerotic lesions is produced locally in the arterial wall after entry of normal LDL from the plasma. This concept is supported by the findings that plasma contains a variety of antioxidants (10) and that Ox-LDL is removed extensively and rapidly from plasma after intravenous injection 11, 12.
However, numerous studies have provided evidence that Ox-LDL is in fact present in the circulating blood in vivo. Thiobarbituric acid-reactive substances (TBARS), a nonspecific measure of lipid peroxidation, are present in plasma, and their concentration is greater in subjects with coronary artery disease than in the controls (13). It is suggested that mildly oxidized LDL (MM-LDL) can circulate in the plasma for a period long enough to enter and accumulate in the arterial wall (14). Autoantibodies reactive with Ox-LDL are present in plasma and increased amounts of IgG autoantibody against oxidized LDL are associated with progression of carotid atherosclerosis (15). There is a hypothesis that anti-Ox-LDL antibodies have a role in the clearance of Ox-LDL from the circulation 16, 17.
Monitoring Ox-LDL levels in human blood would be very useful for studying Ox-LDL functions and atherogenesis and for diagnostic purposes. A simple, rapid and practical assay method to determine the concentration of Ox-LDL in human blood is needed for this purpose. However, the evaluation of Ox-LDL is difficult, given the multiple chemical effects of oxidation. We previously developed an EIA method for measuring oxidatively-modified LDL in human plasma (18). In this EIA method, the mouse monoclonal antibody (MAb) FOH1a/DLH3 was used. This MAb recognized oxidized phosphatidylcholine and showed good reactivity to Ox-LDL but did not react with native LDL or chemically-modified LDL such as acetylated or malondialdehyde-treated LDL 19, 20. This EIA method is useful for measuring Ox-LDL in human plasma, but there are some problems when applying it for widespread clinical use. For instance, when blood samples are to be assayed, the LDL fraction has to be obtained by ultracentrifugation from each sample, which is a time-consuming and laborious job, and some reagents have to be prepared freshly each time for each use. To obtain reproducible and accurate results, the assay conditions have to be rigorously controlled.
Here we describe the development of a new assay, suitable for the measurement of the Ox-LDL in human plasma or serum, which meets the requirement for a routine clinical assay. The clinical utility of this EIA was assessed by measurement of the Ox-LDL in the plasma of normal subjects and patients with coronary heart disease (CHD).
Section snippets
Materials and chemicals
Reagents were obtained from the following sources: bovine serum albumin (BSA) (Miles, Inc., Kankakee, IL, USA); 3,3′,5,5′-tetramethylbenzidine (TMBZ) solution (TMBlue, TSI Co., Milford, MA, USA); anti-human Apo-B goat IgG (Dako Japan Co., Ltd., Kyoto, Japan); N-succinimidyl-6-maleimidohexanoate (Dojindo Laboratories, Kumamoto, Japan); polyethylene glycol 6000 (Nacalai Tesque, Inc., Kyoto, Japan); horseradish peroxidase (HRP) (Toyobo Co., Ltd., Osaka, Japan); Phenyl-sepharose (Pharmacia LKB
Monoclonal antibody
In this study, FOH1a/DLH3 mouse MAb was purified from serum-free culture supernatant by hydrophobic chromatography and gel filtration chromatography. The collected IgM was confirmed to be monomeric and homogeneous by FPLC and SDS-PAGE, respectively (Figure 1). In this EIA, use of the MAb mentioned above produced high specificity and reproducibility.
Enzyme immunoassay for detection of Ox-LDL
A sandwich EIA for plasma Ox-LDL was done with a 96-well microtiter plate as the solid phase, with FOH1a/DLH3 antibody as the capture antibody,
Discussion
The goal of this study was to develop an EIA that met the requirements of routine clinical assay for the measurement of Ox-LDL in blood.
The present EIA is a sandwich-type assay developed using the mouse MAb FOH1a/DLH3, and anti-human Apo-B antibody. The specificity of this EIA is due to the specificity of the antibodies used, especially the monoclonal antibody, FOH1a/DLH3. This antibody shows high specificity for oxidized phosphatidylcholine and does not react with native, acetylated, or
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