Binding properties of antibodies to prothrombin and beta sub(2)-glycoprotein I ( beta sub(2)-GPI) assayed by ELISA and dot blot

• Published in: Clinical and experimental immunology 1999-12, Vol.118 (3), p.480-486
• Main Authors: astiero, R R, Martinuzzo, ME, Carreras, LO
• Format: Article
• Language: English
• Subjects: antiphospholipid syndrome; b2-glycoprotein I; prothrombin
• ISSN: 0009-9104
• DOI: 10.1046/j.1365-2249.1999.01064.x

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta sub(2)-GPI (anti- beta sub(2)-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 beta sub(2)-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- beta sub(2)-GPI by ELISA and dot blot, while 15/25 had anti-II super(ELISA) and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti- beta sub(2)-GPI by ELISA or dot blot, whereas 6/10 had anti-II super(ELISA) (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti- beta sub(2)-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta sub(2)-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta sub(2)-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 mu g/ml) of beta sub(2)-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 beta sub(2)-GPI and prothrombin.