Preparation of a Human Standard for Determination of the Levels of Antibodies to Oxidatively Modified Low-Density Lipoproteins (original) (raw)
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Clinical Biochemistry, 2000
Objectives: To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis. Design and methods: A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL). Results: This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6 -103.8%), simplicity and rapidity (Ͻ4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 Ϯ 2.8 U/mL, mean Ϯ SD) and patients with coronary heart disease (CHD) (19.7 Ϯ 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD. Conclusions: We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.
International journal of clinical & laboratory research, 1993
We have developed a solid-phase enzyme immunoassay for anti-oxidized low-density lipoprotein antibodies. Most sera showed some degree of non-specific binding to plates coated with oxidized low-density lipoprotein and the autoantibodies to oxidized low-density lipoprotein often appeared to have a relatively low affinity. To differentiate between specific and non-specific binding each sample was tested untreated and after absorption with oxidized low-density lipoprotein. The optical densities obtained with dilutions of the absorbed sample were considered to reflect non-specific binding and were subtracted from values obtained with identical dilutions of the unabsorbed sample, to yield corrected values from which the concentrations of anti-oxidized low-density lipoprotein antibody were calculated. Similar absorptions with native low-density lipoprotein and oxidized human serum albumin failed to induce a significant reduction in binding to immobilized oxidized low-density lipoprotein pr...
Auto-antibodies against oxidized low density lipoproteins in systemic diseases
Atherosclerosis, 1995
Oxidation of low density lipoproteins (LDL) obviously plays an important role in the pathogenesis of atherosclerosis. The purpose of the study was to determine whether antibodies against oxidized LDL are associated with coronary artery disease (CAD). We determined the serum levels of antibodies against copper-oxidized LDL by enzyme-linked immunosorbent assay in 58 patients with angiographically verified CAD and 34 controls without CAD. The mean antibody level, expressed in optical density units, was significantly higher in patients than in controls (0.150Ϯ0.088 versus 0.094Ϯ0.054, respectively; Pϭ0.00089). In logistic regression analysis, high antibody level against oxidized LDL was associated significantly with CAD (Pϭ0.0114), independent of age (Pϭ0.00137), gender (Pϭ0.0021), body mass index (Pϭ0.5947), triglyceride concentration (Pϭ0.9813), and total cholesterol-high density lipoprotein (HDL) cholesterol (Pϭ0.0080) group. Similar analysis in nondiabetic subjects (nϭ79) and in men only (nϭ75) showed analogous results, with only minor changes in P values. The antibody level against oxidized LDL differed significantly between nonsmokers and smokers in CAD patients (PϽ0.00197) but not in controls (PϭNS). In addition, the antibody level against oxidized LDL differed significantly between nonsmokers and smokers in subjects with low HDL cholesterol (Յ0.9 mmol/L) but not in subjects with high HDL cholesterol (Ͼ0.9 mmol/L). In conclusion, elevated levels of antibodies against oxidized LDL were associated with CAD. The data suggest that oxidized LDL plays a role in the pathogenesis of atherosclerosis and suggest a protective function for HDL against LDL oxidation.
Clinica Chimica Acta, 2009
Background: Oxidized lipoproteins and antibodies anti-oxidized low-density lipoprotein (anti-oxLDL) have been detected in human plasma and in atherosclerotic lesions. However, the role of these autoantibodies in the maintenance of vascular health or in the pathogenesis of acute vascular insults remains unclear. We examined the relationship of human immunoglobulin G (IgG) anti-oxLDL antibodies with cardiovascular disease risk markers in stable subjects and in patients after an acute coronary syndrome (ACS). Methods: Titers of human anti-oxLDL antibodies were measured in hypertensive subjects in primary prevention (n = 94), without other risk factors, and in individuals after a recent ACS event who also had metabolic syndrome (n = 116). Autoantibodies against copper ion oxidized LDL were measured by enzymelinked-immunosorbent assay. Results: Anti-oxLDL titers were higher in hypertensive patients and these subjects presented lower high sensitivity C-reactive protein (hs-CRP) than those with ACS (p b 0.0001). We found significant correlations between anti-oxLDL and hs-CRP (r=−0.284), body mass index (r=−0.256), waist circumference (r=−0.368), apolipoprotein B (r=−0.191), glucose (r=−0.303), systolic blood pressure (r=0.319), diastolic blood pressure (r= 0.167), high-density lipoprotein cholesterol (r=0.224) and apolipoprotein A1 (r=0.257) (pb 0.02 for all). After multiple linear regression hs-CRP, fasting glucose and waist circumference remained independently and inversely associated with anti-oxLDL. Conclusions: Acute inflammatory and metabolic conditions decrease titers of human antibodies of IgG class against oxidized LDL, and that circulating anti-oxLDL antibodies could be associated with a protective role in atherosclerosis.
Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein
Clinical and Vaccine Immunology, 2005
Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N ε (carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins conta...
Circulation, 2003
Background— Low-density lipoprotein (LDL) can be oxidatively modified by reactive oxygen species, thus generating oxLDL. The latter induce formation of specific antibodies (oxLDLAb), which are detectable in patients with atherosclerosis, in which they might play a pathogenic or a protective role. Thus, we aimed to investigate the association of antibodies with oxidized LDLs (oxLDL) (oxLDLAbs) with coronary artery disease (CAD) and acute coronary syndromes. Methods and Results— In a cross-sectional study of 529 consecutive patients undergoing quantitative coronary angiography for suspected CAD, we measured the titer of IgG oxLDLAbs by ELISA. With regression analysis techniques, we also investigated the determinants of oxLDLAb titer and the association of oxLDLAbs with CAD severity. We found no significant differences of oxLDLAb titer between groups of patients without and with different CAD severity. The oxLDLAb titer was 18.6 enzyme units (EU) (11.5 to 25.7 EU/mL) (mean, 95% CI) in ...
The Journal of Experimental Medicine, 1959
Hemagglutination is a specific and sensitive technique for investigating the purity of lipoproteins and the immunologic relationships between low density lipoprotein fractions. The Sf 10–400 and Sf 3–9 lipoprotein fractions, isolated from human serum by dextran sulfate-density gradient centrifugation procedure and repurified by centrifugation appeared to contain only lipoprotein antigens since these fractions did not stimulate the production of antibodies against other serum proteins. Cross-absorption experiments with lipoproteins carried on "tanned" cells demonstrated that the Sf 3–9 lipoprotein fraction contains all the antigenic components of the Sf 10–400 lipoprotein fraction together with additional antigenic components not found in the Sf 10–400 lipoprotein fraction. Thus Sf 3–9 and Sf 10–400 lipoprotein fractions are immunologically similar but not identical. Low density lipoproteins contain no antigens in common with the high density lipoproteins. An Sf 3–9 antiser...
Antibodies against oxidized LDL--theory and clinical use
Physiological research / Academia Scientiarum Bohemoslovaca, 2001
Modification of low density lipoprotein (LDL) particles due to oxidation, glycation and binding of advanced glycation end-products (AGEs) or malondialdehyde (MDA, a final product of lipid peroxidation) is considered most important in the process of atherogenesis. Oxidatively modified LDL are distinguished by another receptor type, which was discovered on the surface of macrophages and was called the scavenger receptor. Uncontrolled intake of LDL converts macrophages to foam cells; their accumulation under the vascular endothelium is considered as the first stage of atherosclerosis. Oxidation of LDL is a complex process taking place in both the extra- and intracellular space. At the end of this oxidative process, modified LDL particles show chemotactic, cytotoxic and immunogenic properties. Oxidized LDL express a large number of epitopes and cause production of polyclonal autoantibodies against these products, especially against apoB100 modified by MDA and 4-hydroxynonenal. IgoxLDL (...