A major problem for diagnosing the antiphospholipid syndrome (APS) is the high variability between commercial anti-beta(2)glycoprotein I (beta(2)GPI) 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 beta(2)GPI opens up, thereby exposing G40-R43. Objectives: To examine whether suboptimal exposure of epitope G40-R43 explains the variations in results observed between commercial assays. Methods: Two 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 beta(2)GPI irrespective of its conformation. These antibodies were tested in commercial anti-beta(2)GPI assays (A-E). Results: In assay A, both antibodies showed equal reactivity towards beta(2)GPI, indicating that all the beta(2)GPI 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. Conclusions: Exposure of epitope G40-R43 on beta(2)GPI is highly variable between commercial anti-beta(2)GPI assays. As a consequence, patients can be falsely assigned negative in assays characterized by a reduced exposure of G40-R43.