Isolation of functional human coagulation factor V by using a hybridoma antibody.
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 1:10(6) 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 Mr comparable to that of bovine Factor V (330,000). Upon exposure to thrombin, the activity of Factor V increased 53-fold when measured in Factor V-deficient plasma. This increased activity was associated with discrete proteolytic cleavages of the parent molecule.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002999:Clone Cells / Q000276:immunology; D005165:Factor V / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells / Q000276:immunology; D051379:Mice; D009101:Multiple Myeloma; D009374:Neoplasms, Experimental; D011863:Radioimmunoassay; D013154:Spleen; D013917:Thrombin / Q000378:metabolism
D000906:Antibodies; D005165:Factor V; D013917:Thrombin