Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-Beta2-Glycoprotein I IgG ELISAs

Abstract

BackgroundA major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-β2glycoprotein I (β2GPI) assays. Predominantly antibodies reactive against cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I are associated with an increased risk for thrombosis. Upon interaction with anionic surfaces β2GPI opens up, thereby exposing G40-R43.ObjectivesTo examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays.MethodsTwo patient-derived monoclonal antibodies were tested on neutral versus anionic plates. Antibody P1-117 reacts with G40-R43 in the open conformation while P2-6 recognizes β2GPI irrespective of its conformation. These antibodies were tested in commercial anti-β2GPI assays (A–E).ResultsIn assay A, both antibodies showed equal reactivity towards β2GPI, indicating that all the β2GPI exposes G40-R43. In other assays P1-117 displayed lower reactivity than P2-6, demonstrating reduced G40-R43 availability. To exclude influences of other assay features, reactivity was re-examined on plates of assay A and B using the protocol/reagents from each assay. In all combinations, reactivity of both antibodies on a plate was comparable to results obtained with its own protocol/reagents, suggesting that the coating, rather than other assay components, accounts for the observed differences. In two patient cohorts we demonstrated that a number of domain I-reactive samples are missed in assays characterized by a decreased exposure of epitope G40-R43.ConclusionsExposure of epitope G40-R43 on β2GPI is highly variable between commercial anti-β2GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.

Role of anti‐domain 1‐ß2glycoprotein I antibodies in the diagnosis and risk stratification of antiphospholipid syndrome: reply

“…The HemosIL Acustar ab 2 GPI assay (Instrumentation Laboratory, Werfen, Austria) probably detects mainly the antibodies targeting domain I, as determined by the presentation of the protein on the solid phase. Coatings of b 2 GPI on the solid phase differ in exposure of the domain I G40-R43 epitope, and may therefore influence the performance of the assays [6,7]. We previously demonstrated good sensitivity of this ab 2 GPI CIA for aD1 antibodies.…”

“…Whereas we and Zhang et al used a commercially available automated chemiluminescence assay (CIA) (QUANTA Flash), de Laat et al performed analyses with an inhouse designed ELISA [4]. After all, detection of aD1 antibodies depends on the coating of b 2 GPI on the solid phase, as availability of the immunogenic epitope is crucial [6]. To our knowledge, no published data are available on a head-to-head comparison between the commercial and in-house assay for aD1 antibodies.…”

Laboratory criteria for antiphospholipid syndrome: communication from the SSC of the ISTH

“…In external quality control exercises and using human monoclonal antibodies with different reactivity towards b2GPI discriminating detection of open and closed confirmation of b2GPI, differences were demonstrated in commercially available ab2GPI IgG ELISA assays, as well as in automated systems [21][22][23].…”

Anti-beta-2 glycoprotein I epitope specificity: from experimental models to diagnostic tools

“…The reactivity against DI was associated anti-b2GPI antibodies PL Meroni with the full-blown APS clinical picture and specifically with the positivity for LA, suggesting a higher diagnostic/prognostic value for these aPL. 19,20 The main DI epitope was reported to involve arginine 39-arginine 43, aspartic acid 8-aspartic acid 9, and possibly the interlinking region between DI and DII, with R39 being the most important residue. 21,22 The epitope in the circular and the S-shape forms of the molecule is not available for autoantibody binding but can be exposed in the open J configuration.…”