Antibodies to β2-Glycoprotein-I: Urea Resistance, Binding Specificity, and Association with Thrombosis

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Abstract

The aim of the present study was to evaluate the urea resistance and binding characteristics of anti-β2-glycoprotein I (anti-β2GPI) antibodies using standard anticardiolipin (aCL) and anti-β2GPI enzyme immunosorbent assays (ELISAs). Sera from patients with antiphospholipid syndrome (APS) (n = 22) and non-APS (n = 24), positive in a standard aCL ELISA, were tested in an anti-β2GPI ELISA performed in polystyrene-irradiated ELISA plates. Urea resistance aCL and anti-β2GPI ELISAs were performed by measuring the ability of antibodies to recognize antigen in the presence of 2 M urea. The serum dilution after urea treatment (D) expressed as a percentage of the serum dilution without urea treatment (Do) corresponding to the same optical density was defined as residual activity (RA = 100 D/Do). The higher the RA, the higher the resistance of the antibodies to urea. APS compared to non-APS sera had higher aCL binding (absorbance values ranging between 0.180 and 1.400; median, 0.717 vs 0.120-1.273; median, 0.250, respectively; P < 0.004). Six APS patients' sera had low aCL levels but they expressed RA ≥30%. Anti-β2GPI antibodies were detected in 15 of 22 APS vs 3 of 24 non-APS patients (P < 0.03); RA ≥30% was detected in 15 of 22 APS vs 1 of 23 non-APS patients (P < 0.004). Using a CL affinity column, antibodies were purified from three APS anti-β2GPI negative and three non-APS anti-β2GPI-positive patients and tested in a aCL ELISA, using highly purified bovine serum albumin (BSA) as a blocking agent (modified ELISA); reactivity was not detected in two APS and one non-APS sera. On the contrary, the reactivity of the purified antibodies was high when β2GPI was incubated with CL in the ELISA plates; thus some anti-β2GPI negative sera from APS patients recognized the CL/β2GPI complex, rather than CL or β2GPI alone. In conclusion, anti-β2GPI antibodies are common in the APS patients, but a number of such patients recognize the CL/β2GPI complex and not CL or β2GPI. Antibodies to either β2GPI or the CL/β2GPI complex derived from APS sera present a high resistance to urea. Anti-β2GPI antibodies of low urea resistance exist in a minority of non-APS patients with autoimmune disease.

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