Variability in Exposure of Epitope G40-R43 of Domain I in Commercial Anti-β₂-Glycoprotein I IgG Elisas Influences the Diagnosis of the Antiphospholipid Syndrome

Diagnosis of the antiphospholipid syndrome (APS) depends on the detection of autoantibodies against phospholipid-bound β₂-glycoprotein I (β₂GPI), the most prominent antigen in APS. One of the main problems faced is the high variability observed between different commercially available anti-β₂GPI assays. Anti-β₂GPI antibodies constitute a heterogeneous population, but predominantly antibodies reacting to a cryptic epitope Glycine40-Arginine43 (G40-R43) in domain I of β₂GPI are associated with a strong risk to develop thrombosis. b₂GPI is present in blood in a native conformation (either circular or S-shaped). After interaction with anionic surfaces it opens up (J-shaped conformation), resulting in exposure of the epitope G40-R43 in domain I. It is therefore important that in diagnostic assays b₂GPI is presented in the open conformation enabling antibodies to react with the G40-R43 epitope. We hypothesize that the high variability between different commercial anti-b₂GPI ELISA assays arise from variation in exposure of the G40-R43 epitope of b₂GPI.

Two patient-derived monoclonal antibodies P2-6 and P1-117 were tested for their reactivity towards β₂GPI in different conformations. Using both antibodies, we compared exposure of epitope G40-R43 on β₂GPI in five different commercial anti-β₂GPI IgG assays. 10 patient samples selected for their low to high positivity towards epitope G40-R43, were tested in two anti-β₂GPI IgG assays.

Using neutral versus anionic ELISA plates, we have shown that antibody P1-117 specifically reacts with epitope G40-R43, exposed only in the open conformation, while antibody P2-6 recognizes β₂GPI irrespective of its conformation (Fig. 1). In one of the tested anti-β₂GPI assays, both antibodies showed equal reactivity towards β₂GPI, indicating that all the coated β₂GPI exposes epitope G40-R43. In this assay, all ten anti-domain I positive patients tested positive. In other assays P1-117 displayed lower reactivity towards β₂GPI than P2-6, demonstrating a reduced exposure of G40-R43. Only 3 out of the 10 patients tested positive in such assay, illustrating that a significant number of patients can be falsely assigned negative in assays characterized by a reduced exposure of epitope G40-R43.

The exposure of epitope G40-R43 on β₂GPI is highly variable in commercial anti-β₂GPI assays, and influences the diagnosis of APS. Our results have major implications for the diagnosis of APS, as it provides suitable controls to ensure sufficient exposure of the G40-R43 epitope or suggests the development of alternative assays coating only domain I of β₂GPI.

Fig. 1.

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Binding sites of antibodies P1-117 and P2-6 on the J-shaped and native (circular or S-shaped) conformation of b₂GPI

Fig. 1.

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Binding sites of antibodies P1-117 and P2-6 on the J-shaped and native (circular or S-shaped) conformation of b₂GPI

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No relevant conflicts of interest to declare.