Immunoelectrophoresis and ELISA techniques for assay of plasma beta 2 glycoprotein-1 and the influence of plasma lipids
Mcnally, T.; Mackie, I.J.; Isenberg, D.A.; Machin, S.J.
Thrombosis Research 72(4): 275-286
1993
ISSN/ISBN: 0049-3848 PMID: 8303667 Document Number: 415187
beta-2 glycoprotein-I (beta-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 beta-2GPI. Western blotting and crossed immunoelectrophoresis (CIE) of plasma demonstrated free beta-2GPI and beta-2GPI complexed with unidentified plasma constituents. The free and complexed forms were not distinguished in immunoelectrophoresis assays, allowing measurement of total beta-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 coefficients 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 beta-2GPI used to standardize the assays was shown to have subfraction selectivity and different calibration values were obtained for pooled normal plasma by the unmodified IEP (242 mg/l) and modified IEP and ELISA (201 mg/l). We have also investigated the influence of some pre-test variables on beta-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 beta-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 beta-2GPI in healthy subjects.