USE OF MUTATIONS IN DOMAIN 1 AND DOMAIN 4 OF ß2GPI TO DETERMINE FINE ANTIGENIC SPECIFICITY OF ANTIPHOSPHOLIPID AUTOANTIBODIES

G. Michael Iverson1, Eiji Matsuura2, Edward J. Victoria 1, Keith A. Cockerill1 and Matthew D. Linnik1

1 La Jolla Pharmaceutical Company, 6455 Nancy Ridge Drive. San Diego, California
2 Okayama University, Japan

ß2-glycoprotein I (ß2GPI) is a plasma protein comprised of five domains that has been shown to play an important role in the antigenic specificity of antiphospholipid autoantibodies. We used error-prone PCR to generate a library of domain 1 single point-mutants expressed on phage. Ten mutations were expressed as single point mutations on full-length ß2GPI in insect cell cultures. These 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. Two recombinant ß2GPI proteins, with mutations in domain 4, were also examined. One called Triple, had D193, D222 and E228 replaced by V. The other had a single amino acid substitution where W235 was replaced with L. The results show that autoantibodies bind very poorly to these domain 4 mutants when tested by direct binding ELISA, on polyoxygenated plates. On the other hand both are no less effective than wild type ß2GPI from inhibiting the same autoantibodies binding in a competitive inhibition assay. Finally, we show that adsorption with immobilized domain 1 completely removes all anti-ß2GPI reactivity when tested on cardiolipin coated plates. Taken together these data show that the autoantibodies from patients with APS definitely react with domain 1 of ß2GPI.

Presented at the
3rd International Congress on Autoimmunity
Geneva, Switzerland
February 20-24, 2002.