Anti-β2GPI IgG display a broad reactivity against different β2GPI domains beyond domain 1: results from the APS ACTION and multi-center Italian cohorts

Background Anti-β2glycoprotein I (β2GPI) antibodies are the hallmark of the antiphospholipid syndrome (APS). β2GPI consists of five domains, DI-DV. While DI is the primary target, the clinical relevance of antibodies against other domains remains uncertain. We analyzed two large anti-β2GPI IgG positive cohorts, to investigate whether different domain binding affects anti-β2GPI IgG assay testing and APS diagnosis. Methods The presence of anti-DI and anti-DIV/DV antibodies (by chemiluminescence (CLIA) and in-house ELISA, respectively) was searched in an APS ACTION (n.191) and Italian validation (n.105) cohorts. In the latter, we detected anti-β2GPI IgG reactivity by four assays (in-house and commercial ELISA, CLIA, and fluorescence enzyme immunoassay), and used a modified anti-β2GPI IgG in-house ELISA with recombinant single-domain-lacking β2GPI variants to evaluate domain-dependent reactivity. Results We confirmed anti-DI, anti-DIV/DV β2GPI IgG direct reactivity in classified and non-classifiable APS in the two cohorts, and found anti-DI/anti-DIV/DV double negative (38/191, 29/105) and anti-DIV/DV single-positive (6/191, 9/105) samples. Anti-β2GPI IgG discordant samples by the four methods had the highest presence of anti-DIV/DV single-positives and anti-DI/anti-DIV/DV double-negatives, compared to four-method concordant samples: 16% vs 2% and 45% vs 11% (p <0.0001), respectively. The single-domain molecule-based assay showed that the APS serum samples depended mainly on DI, DV, and DII, slightly on DIV, and not at all on DIII. Conclusions Serum IgG from both classified and non-classifiable APS may react with other β2GPI domains than DI and DIV-V. Anti-β2GPI domain selectivity can explain discordant results among diagnostic assays.