To ascertain the heterogeneity of anti-prothrombin antibodies (aPT), we compared three in-house aPT ELISAs: A) medium binding plates, phosphatidylserine, prothrombin, Tris-buffered saline, calcium; B) high binding plates, prothrombin, Tris-buffered saline; and C) high binding plates, prothrombin, PBS. One serum, exhibiting high positive aPT in all three ELISAs, was selected as the calibrator. Sera from 47 patients (41 with SLE, 4 with pAPS, 2 with arterial thromboses) were tested for IgG and IgM aPT.
The results showed six different patterns: 1) similar results were obtained with A, B and C (similar analytical sensitivity); 2) similar results were obtained with A and B while C showed lower analitycal sensitivity; 3) B and C seemed analytically less sensitive than A; 4) A and B were analytically less sensitive than C; 5) A showed very low analytical sensitivity; 6) A and C showed lower analytical sensitivity than B.
The analysis of all the presented patterns showed noncomparable results of the three ELISAs. In our experiments, prothrombin was recognized by relevant antibodies when the protein was bound to phosphatidylserine-coated microtiter plates using calcium ions or when it was bound to high binding plates with or without calcium ions. Nevertheless, detected aPT values were not of the same fine specificity. Different binding conditions for prothrombin exposed different epitopes, resulting in the detection of various subgroups of aPT.