Isolation of functional human coagulation factor V by using a hybridoma antibody.

Abstract

Spleen cells obtained from mice immunized with partially purified human coagulation factor V were fused with NS-1 mouse myeloma cells, and hybrids were selected. Culture media were screened for anti-Factor V activity, and an antibody-positive clone was obtained and passaged as an ascites tumor in mice. The ascitic fluid from the hybridoma-bearing mouse could be diluted 1:106 before losing reactivity in an anti-factor V radioimmunoassay. When immobilized on agarose, the monoclonal antibody quantitatively removed factor V activity from human plasma. Factor V activity could be eluted with 1.2 M NaCl at pH 6.5. Homogeneous factor V was isolated by chromatography of barium citrate-adsorbed, polyethylene glycol 6000 precipitated plasma on the antibody column followed by chromatography on phenyl-Sepharose. The isolated factor V exhibited a single band upon gel electrophoresis in sodium dodecyl sulfate with an apparent MW comparable to that of bovine factor V (330,000). Upon exposure to thrombin, the activity of factor V increased 53-fold when associated with discrete proteolytic cleavages of the parent molecule.