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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 (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.

SUBMITTER: Chinnaraj M

PROVIDER: S-EPMC8455811 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Rational Laboratory Diagnostics of Antiphospholipid Antibodies: Anti-Cardiolipin, Anti-?2-Glycoprotein I, Anti-Prothrombin and Anti-Annexin V Antibodies.

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Associations with thrombosis are stronger for antiphosphatidylserine/prothrombin antibodies than for the Sydney criteria antiphospholipid antibody tests in SLE.

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Project description:Abstract Objective Real-time reverse transcription-polymerase chain reaction is the gold standard for the diagnosis of COVID-19, but it is necessary to utilize other tests to determine the burden of the disease and the spread of the outbreak such as IgG-, IgM-, and IgA-based antibody detection using enzyme-linked immunosorbent assay (ELISA). Materials and Methods We developed an indirect ELISA assay to quantitatively measure the amount of COVID-19 IgG, IgM, and IgA antibodies present in patient serum, dried blood, and plasma. Results The population cutoff values for positivity were determined by receiver operating characteristic curves to be 1.23 U/mL, 23.09 U/mL, and 6.36 U/mL for IgG, IgM, and IgA, respectively. After albumin subtraction, the specificity remained >98% and the sensitivity was 95.72%, 83.47%, and 82.60%, respectively, for IgG, IgM, and IgA antibodies to the combined spike subunit 1 receptor binding domain and N proteins in serum. Plasma and dried blood spot specimens were also validated on this assay. Conclusion This assay may be used for determining the seroprevalence of SARS-CoV-2 in a population exposed to the virus or in vaccinated individuals.

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