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.