Binding properties of antibodies to prothrombin and β2-glycoprotein I (β2-GPI) assayed by ELISA and dot blot

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards β2-GPI (anti-β2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of β2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-β2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-β2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-β2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to β2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with β2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 μg/ml) of β2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on β2-GPI and prothrombin

Lipid peroxidation as risk factor for endothelial dysfunction in antiphospholipid syndrome patients

The aim of this study was to evaluate oxidative stress markers and it relations to endothelial damage as risk factor for thrombosis in patients with primary (PAPS) and secondary (SAPS) antiphospholipid syndrome (APS) in correlation to traditional risk factors. Flow-mediated (FMD) and nitroglycerine (NMD)-induced dilation of the brachial artery were studied in 140 APS patients (90 PAPS, 50 SAPS) and 40 controls matched by age, sex, and conventional risk factors for atherosclerosis. Markers of oxidative stress, lipid hydroperoxydes (LOOH), advanced oxidation protein products (AOPP), total sulfhydryl groups (tSHG), and paraoxonase 1 activity (PON1) were determined by spectrophotometric method. Oxidative stress dominates in APS patients. LOOH and AOPP correlate to lipid fractions (p lt 0.05), unlike PON1, tSHG that correlated to antiphospholipid antibody positivity (p lt 0.05). FMD was lower in APS patients comparing to controls (p lt 0.001). Cholesterol is independent variable for FMD impairment in control group (p = 0.011); LOOH in PAPS (p = 0.004); LOOH, aCL, and triglycerides in SAPS patients (p = 0.009, p = 0.049, and p = 0.012, respectively). Combined predictive of aCL and LOOH is better for FMD impairment than LOOH alone in both PAPS and SAPS patients (AUC 0.727, p = 0.001, 95 % CI 0.616-0.837 and AUC 0.824, pE,0.001, 95 % CI 0.690-0.957, respectively). Lipid peroxidation is independent predictor for endothelial dysfunction in APS patients. We demonstrated synergistic effect of aCL and LOOH as risk for endothelial impairment in both PAPS and SAPS patients