THE ORIENTATION OF b2-GLYCOPROTEIN I (b2-GPI) ON THE PLATE IS IMPORTANT FOR THE BINDING OF ANTI-b2-GPI AUTOANTIBODIES BY ELISA

G. Michael Iverson, Eiji Matsuura,* Edward Victoria, Keith A. Cockerill and Matthew Linnik
La Jolla Pharmaceutical Co. 6455 Nancy Ridge Drive, San Diego, CA, *Department of Cell Chemistry, Okayama University Graduate School of Medicine and Dentistry Okayama, Japan

b2-Glycoprotein I (b2-GPI) is a plasma protein comprised of five complement control protein domains (CCP or "sushi") that has been shown to play an important role in the antigenic specificity of antiphospholipid autoantibodies. These autoantibodies are associated with thrombosis and recurrent fetal loss in humans. We have analysed the ELISA conditions used to measure these autoantibodies. We used two recombinant b2-GPI proteins, with mutations in domain 4, another with a single point mutation in domain 1 plus wildtype b2-GPI. One called Triple, had D193, D222 and E228 replaced by V. Another had a single amino acid substitution where W235 was replaced with L. Yet another had a single amino acid substitution where R43 was replaced with G. The results show that autoantibodies bind very poorly to these two domain 4 mutants or to the domain 1 mutant when tested by direct binding ELISA, on polyoxygenated plates. On the other hand, inhibition profiles of both domain IV mutants were different from that of domain 1 mutant on autoantibodies binding to the wild type b2-GPI solid phase. This observation suggests that the orientation of the b2-GPI on the plate is important for the binding of autoantibodies.

Presented at the
10th International Congress on APS
Sicily, Italy
September 29-October 3, 2002