A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis

Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R(2 =) 0.85) but not with aPT-A (R(2 =) 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.