Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes.
Two different monoclonal antibodies against the heparin-dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas. One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for APC-PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor. The purified inhibitor formed SDS stable complexes with activated protein C and urokinase that could be identified in immunoblots using specific antibodies.
Laurell
M
M
Department of Clinical Chemistry, Malmö General Hospital, Sweden.
Carlson
T H
TH
Stenflo
J
J
D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D011486:Protein C / Q000037:antagonists & inhibitors*
D000911:Antibodies, Monoclonal; D011486:Protein C; D006493:Heparin