Advances in understanding what we measure when detecting anticardiolipin autoantibodies.

Understanding so-called anticardiolipin autoantibodies has been the focus of significant interest due to their accepted role in the pathology of antiphospholipid syndrome (APS). How these antibodies contribute to thrombosis, stroke and recurrent fetal loss is not clearly understood. A precise determination of the antigenic epitope(s) recognized by anticardiolipin autoantibodies will contribute to an understanding of their role in this complex disease; however, there have been many conflicting reports regarding these epitope(s), and many of the apparent contradictions arise from the assay systems that have been used. In this review, we attempt to highlight what we believe are the salient points on this issue. We present further evidence supporting our hypothesis that most of these autoantibodies recognize epitope(s) located on domain 1 (DI) of beta2-glycoprotein 1 (beta2GPI).

In vivo characterization of bioconjugate B cell toleragens with specificity for autoantibodies in antiphospholipid syndrome.

This study investigated the use of well-defined bioconjugate molecules to suppress antigen-specific B cell responses to domain I (DI) of human beta(2)-glycoprotein I (beta(2)GPI) in rats. DI is the dominant target of pathogenic autoimmune antibodies in patients with antiphospholipid syndrome (APS), a disease characterized by antibody-mediated thromboembolic events. Rats primed with DI conjugated to keyhole limpet hemocyanin (DI-KLH) were rendered tolerant to subsequent antigen challenge by treatment with multivalent conjugates of DI. Antibodies to DI were suppressed 89-96% with intravenous doses of 500 micro g, and reductions were paralleled by decreases in splenic antigen-specific antibody-forming cells (AFC). Suppression was achieved with a variety of conjugates having two to four copies of DI and circulating half-lives of 2.6-8.7 h. Antibodies to KLH were not suppressed, indicating the specificity of the approach. These results establish the basis for further development of therapeutic B cell toleragens to suppress pathogenic antibodies in APS and other autoimmune diseases.

Use of single point mutations in domain I of beta 2-glycoprotein I to determine fine antigenic specificity of antiphospholipid autoantibodies.

Autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) appear to be a critical feature of the antiphospholipid syndrome (APS). As determined using domain deletion mutants, human autoantibodies bind to the first of five domains present in beta(2)GPI. In this study the fine detail of the domain I epitope has been examined using 10 selected mutants of whole beta(2)GPI containing single point mutations in the first domain. The binding to beta(2)GPI was significantly affected by a number of single point mutations in domain I, particularly by mutations in the region of aa 40-43. Molecular modeling predicted these mutations to affect the surface shape and electrostatic charge of a facet of domain I. Mutation K19E also had an effect, albeit one less severe and involving fewer patients. Similar results were obtained in two different laboratories using affinity-purified anti-beta(2)GPI in a competitive inhibition ELISA and with whole serum in a direct binding ELISA. This study confirms that anti-beta(2)GPI autoantibodies bind to domain I, and that the charged surface patch defined by residues 40-43 contributes to a dominant target epitope.

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.