Electroimmunoassay of prothrombin

Electroimmunoassay of normal (10-Gla) and Gla-deficient prothrombins containing 0 to 9 Gla (gamma-carboxyglutamyl) residues performed in EDTA against anti- (normal) prothrombin showed that each of the Gla-deficient proteins contained as much antigenic activity as does the normal molecule. In the presence of Ca2+, however, normal prothrombin appeared to show the least while 0- to 2-Gla variants, the most activity. This anomaly arises from the presence of antibodies (Ca-IIAb) which form antigen-antibody (Ag-Ab) complexes when the conformation of the antigen is stabilized in Ca2+. For example, upon fractionation of the antisera and mixing Ca-IIAb (bound to 10-Gla prothrombin, Affi-gel column and eluted by replacing Ca2+ with EDTA) with those reacting with prethrombin1 (non-Gla portion of the molecule), rocket heights in Ca2+ decreased the most with 10- and 9-Gla (32%), followed by 13% with 8-Gla, 2% with 7-Gla and none with 2-Gla prothrombins--indicating that Ca-IIAb do cross react with 7-, 8- and particularly 9-Gla prothrombins. This was also confirmed by the fact that anti 7-Gla but not anti 0-Gla sera contained some antibodies which reacted only in the presence of Ca2+.