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

Abstract

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 (R2 = 0.85) but not with aPT-A (R2 = 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.

Added value of antiphosphatidylserine/prothrombin antibodies in the workup of thrombotic antiphospholipid syndrome: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies

“…These results do not support the idea of using aPS/ PT as surrogate marker for LAC as some groups have previously suggested. 20,40,52 53 Assessment of aPL profiles is important for risk stratification purposes, especially in APS and SLE patients. 2,[54][55][56] LAC is considered the strongest independent risk factor for thrombosis of all criteria aPL.…”

“…Prospective studies should be performed to investigate the clinical risk associated with double aPL positivity without LAC positivity and presence or absence of aPS/PT positivity. Research efforts may lead to alternative aPS/PT or anti‐prothrombin assays with better performance and more added value 53 …”

Noncriteria antiphospholipid antibodies in antiphospholipid syndrome

“…Using a biotinylated prothrombin variant looked promising to apply in other types of solid-phase assays than ELISA, to facilitate the detection of aPT by automated systems. 58 Also, a particle-based multi-analyte technology measuring a spectrum of antibodies including aPS/PT has been evaluated with promising results. 59 However, in most of the studies, the only commercially available ELISA is frequently used which is an advantage for the comparability of the studies.…”

“…Recently, a novel ELISA has been described that identified specific aPT that correlated well with aPS/PT, including potential benefit in APS patients. Using a biotinylated prothrombin variant looked promising to apply in other types of solid‐phase assays than ELISA, to facilitate the detection of aPT by automated systems 58 . Also, a particle‐based multi‐analyte technology measuring a spectrum of antibodies including aPS/PT has been evaluated with promising results 59 .…”

Solid Phase Assays for Antiphospholipid Antibodies

“…68 Recently reported, a newly developed ELISA looks promising to detect pathogenic antiprothrombin antibodies by coating the ELISA plate with a novel prothrombin variant. 69 Although searching for aPS/PT in daily practice is not recommended yet, in some conditions aPS/PT might help, especially in confirming the risk. Adding aPS/PT to the triple positive profile, further consolidates the diagnosis of APS.…”