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

Isotype distribution and clinical relevance of anti-β2-glycoprotein I (β2-GPI) antibodies: importance of IgA isotype

The aim of this study was to evaluate the prevalence of IgG, IgA and IgM anti-β2-GPI antibodies in anti-phospholipid syndrome (APS), and to establish the clinical significance of IgA type antibodies compared with the other isotypes. Anti-β2-GPI antibodies were measured in the sera of 70 patients by solid-phase enzyme immunoassay in γ-irradiated polystyrene plates coated with human purified β2-GPI. Thirty-three out of the 70 patients were classified as having APS: three of them had primary, and 30 had secondary APS related to systemic lupus erythematosus (SLE). The remaining 37 patients had SLE without APS. Anti-β2-GPI antibodies of IgG, IgA and IgM isotypes were present in 84.8%, 59.3% and 51.5% of patients with APS. Both the frequency and the level of each isotype were significantly higher in patients with APS. This association was very strong for IgA (P = 0.0004 for the antibody frequency and P < 0.0001 for the antibody level), as well as for IgG type antibodies (P < 0.0001 and P < 0.0001), whereas it was weaker for IgM (P = 0.01 and P = 0.04). A strong relationship was demonstrated between increased IgA anti-β2-GPI antibody levels and a history of venous thrombosis, thrombocytopenia, heart valve disease, livedo reticularis and epilepsy. IgG anti-β2-GPI antibodies were associated with the presence of lupus anticoagulant (LA) in addition to the main features of APS. However, antibodies of IgM isotype were related only to thrombocytopenia and heart valve disease. We recommend the evaluation of anti-β2-GPI antibodies of IgA isotype in addition to IgG in patients with clinical suspicion of APS