Factor V antigen in the plasma of the proband and family members.

(A) Measurement of factor V antigen by 3 different sandwich ELISAs. The following capture antibodies were used: a polyclonal anti–factor V antibody (total factor V antigen); a monoclonal antibody against an epitope on factor V B-domain that detects the N-terminal portion of the molecule containing factor V HC plus the connecting region (FVHC antigen); and a monoclonal antibody against an epitope on factor V light chain (FVLC antigen). The reference interval obtained by Montefusco et al12 was used for comparison. (B) The detection of factor V by Western blotting. SDS-PAGE was carried out in 5% gel. The sample loaded onto the gel contained 0.625 μL normal (lane 1) and 12.5 μL patient (lane 2) plasma or factor V immunoprecipated from 2.5 μL normal plasma (lane 3) and 250 μL patient's (lane 4) plasma. Biotinylated polyclonal antibody against factor V and Vectastain ABC kit were used for the detection of factor V in plasma samples (lanes 1,2), while the blots of immunoprecipitates (lanes 3,4) were developed by monoclonal anti–factor V B-domain antibody and peroxidase-labeled antimouse IgG. Arrows indicate the position of M_r marker proteins; arrowheads point to intact factor V molecule; and double arrowhead points to the faint band representing truncated factor V present in the patient's sample. In the immunoprecipitate from normal control plasma, some breakdown product of factor V could also be seen.