Detergent and antigen fragility affect the ELISA for measurement of anti-prothrombin autoantibodies

Objective. Same investigators have reported that anti-prothrombin autoantib odies (aPT) in lupus anticoagulant positive sera were detectable by ELISA. Discrepancies in aPT ELISA were observed by some investigators. To clarify this situation, we tested the binding of aPT positive sera to purified prot hrombin under various conditions. Methods. Wt performed aPT ELISA under different conditions. The variables w e tested were: ELISA plate (untreated or gamma irradiated polystyrene plate s), buffer (phosphate buffered saline or Tris buffered saline), detergent ( presence or absence of Tween-20), and antigen condition (intact or fragment ed prothrombin). Results, Anti-PT from patients with lupus or antiphospholipid syndrome were similarly bound to prothrombin with both buffers. Addition of Tween-20 to the buffer increased reactivity in the irradiated plate assay, but decrease d reactivity in the untreated plate assay. Reactivities in 90% of lupus ser a were decreased by the use of fragmented prothrombin. In contrast, the rea ctivity of serum from a healthy subject was remarkably increased by antigen fragmentation. Conclusion. The discrepant ELISA results in measurement of aPT in the vario us reports may have been due to the use of detergent in the buffer and cond ition of the prothrombin used as antigen. In our experiments the best ELISA condition for measurement of aPT was achieved using buffer with Tween-20 d etergent, with prothrombin directly coated onto irradiated polystyrene plat es.