Immunoelectrophoresis and ELISA techniques for assay of plasma β2 glycoprotein-i and the influence of plasma lipids

Abstract

β 2 glycoprotein-I ( β 2GPI) has been identified as a cofactor for the binding of some antiphospholipid antibodies to anionic phospholipids and has been demonstrated to possess anticoagulant properties in vitro. We have investigated Laurell rocket immunoelectrophoresis (IEP) and ELISA techniques for measurement of β 2GPI. Western blotting and crossed immunoelectrophoresis (CIE) of plasma demonstrated free β 2GPI and β 2GPI complexed with unidentified plasma constituents. The free and complexed forms were not distinguished in immunoelectrophoresis assays, allowing measurement of total β 2GPI. Standard and detergent modified IEP and ELISA techniques were compared: significant correlation was demonstrated between unmodified and detergent modified IEP, detergent modified IEP and ELISA and unmodified IEP and ELISA. The intra-assay co-efficients of variation (CVs) of the unmodified and modified IEPs and ELISA were 7.5, 3.7 and 7.9%. Inter-assay CVs determined for the modified IEP and ELISA were 5.8 and 9.1% respectively. The purified β 2GPI used to standardise the assays was shown to have subfraction selectivity and different calibration values were obtained for pooled normal plasma by the unmodified IEP (242 mg/I) and modified IEP and ELISA (201 mg/I). We have also investigated the influence of some pre-test variables on β 2GPI levels and shown that heparin, citrate or EDTA plasma and serum samples are suitable for assay of this glycoprotein and that levels are unaffected by repeated freeze/thawing. The influence of plasma lipids on β 2GPI measurement was also examined and we demonstrated no significant differences between pre and postprandial samples, which suggests that fasting status is not an important consideration for assay of β 2GPI in healthy subjects.