The orientation of beta2GPI on the plate is important for the binding of anti-beta2GPI autoantibodies by ELISA.
(Beta2-glycoprotein I (beta2GPI) is a plasma protein that plays an important role in the antigenic specificity of antiphospholipid autoantibodies (aPL). These antibodies are associated with an increased risk for thrombosis and recurrent foetal loss in humans. Crystallographic analysis of beta2GPI showed that its five complement control protein (CCP) or 'sushi' domains are arranged in an elongated, fish-hook shape; yet the domain-specific location of epitopes recognized by these autoantibodies has remained the subject of considerable controversy. Investigators have used different forms of recombinant beta2GPI and different ELISA methods to obtain conflicting results. One group mapped autoimmune epitopes to domain I using deletion mutants of beta2GPI in a competitive inhibition ELISA on NUNC Maxisorp microplates. Another group mapped epitopes to domain IV using beta2GPI with mutations in domain IV in a direct binding ELISA on polyoxygenated microplates. In an effort to resolve these discrepancies, a collaboration between the groups compared wildtype beta2GPI with domain IV mutants in both types of ELISA. Autoantibodies bound very poorly to domain IV mutants coated on polyoxygenated plates, yet they bound very well to the same mutants coated on NUNC Maxisorp plates. The amount of protein adsorbed on to both types of plates was similar. In the competitive inhibition ELISA, no difference could be detected between wildtype beta2GPI and domain IV mutants. These results strongly suggest that the orientation of beta2GPI on the microplate, and not necessarily the lateral density, plays the predominant role in the binding of autoantibodies.
Iverson
G Michael
GM
La Jolla Pharmaceutical Co., 6455 Nancy Ridge Drive, San Diego, CA 92121, USA. mike.iverson@ljpc.com
Matsuura
Eiji
E
Victoria
Edward J
EJ
Cockerill
Keith A
KA
Linnik
Matthew D
MD
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D016736:Antiphospholipid Syndrome / Q000175:diagnosis / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D018604:Epitope Mapping; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D053482:beta 2-Glycoprotein I
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D006023:Glycoproteins; D011994:Recombinant Proteins; D053482:beta 2-Glycoprotein I