G. Michael Iverson, Eric M. Smith, Edward J. Victoria, David M. Marquis, Keith A. Cockerill and Matthew D. Linnik.

ß2-glycoprotein I (ß2GPI) 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. Previous work from this laboratory using whole domain deletion mutants of recombinant ß2GPI expressed in insect cells showed that domain 1 is important for the binding of human affinity-purified antiphospholipid antibodies.1 Antiphospholipid antibodies also bind preferentially to domain 1 expressed as a pIII fusion protein on filamentous phage. We used error-prone PCR to generate a library of domain 1 single point-mutants expressed on phage. A panel of affinity purified antiphospholipid autoantibodies were used to screen these phage. Ten mutations, some that had no effect and some that reduced antibody binding, were then expressed as single point mutations on full-length ß2GPI in insect cell cultures. These mutants were then compared, in competitive inhibition ELISAs, with wild type ß2GPI. The results show that amino acids in positions 19, 40 and 43 are important for binding these autoantibodies. The results further confirm the antigenic importance of domain 1 and suggest that autoantibodies from different patients may recognize similar but not identical epitopes.

Presented at the
9th International Symposium on Antiphospholipid Antibodies
September 12-16, 2000
Tours, Loire Valley, France

Also presented at the
26th Annual Conference of the La Jolla Immunologists
La Jolla, California, USA