Heparin cofactor II: purification and antibody production

Heparin cofactor II (HCII) was purified from plasma to homogeneity. The procedure includes adsorption with (A1)OH3, fractionation with polyethylene glycol 6000, chromatography on QAE-Sephadex A-50, on heparin-Sepharose 4B and on Sephadex G-150. QAE-Sephadex A-50 chromatography provides a good separation of HCII from antithrombin III (AT) and most contaminants having a heparin affinity similar to that of HCII. HCII is eluted at 0.28 M NaCl from the heparin-Sepharose column. After gel filtration on G-150, contaminating AT was removed by immunoadsorption. Purified HCII shows an apparent Mr of 66,500 daltons as analyzed on SDS-polyacrylamide gel and 62,100 daltons by ultracentrifugation. Antibodies to HCII were raised in rabbits. Former antisera mostly directed to a contaminating protein were used to remove it from the HCII preparation. Antibodies to HCII were made monospecific by immunoadsorption on HCII-free plasma linked to Sepharose 4B. Since many functional AT assays have neglected the presence of HCII in plasma, antibodies to HCII using as immunoadsorbent will provide a more specific test for AT.