Mice were immunized with factor VIII complex and boosted with partially purified VIII coagulant (VIII:C). These mice produced antisera which caused inhibition of VIII: C activity in clotting assays. Spleen cells from the antibody positive mice were fused with NS-1 plasmacytoma cells, aliquoted into 96 cultures, and 68 culture wells positive for hybrid cell formation were assayed for anti-VIII:C antibodies. An immunoradiometric assay using 125[I]-labelled human factor VIII:C inhibitor was used to identify anti-VIII:C antibodies. Twelve hybrid supernatants mediated significant binding of the radiolabelled probe in the presence of partially purified VIII:C. Of these, 8 were shown to be positive through the binding of porcine Willebrand factor, rather than direct factor VIII:C binding. Three hybrid supernatants bound factor VIII:C from a preparation of dissociated low molecular weight coagulant, could not be dissociated from the antigen with 0.25 M calcium chloride and bound no detectable porcine Willebrand factor. One of these hybrids was subcloned and grown as an ascitic tumor in mice. After labelling with 125[I], the monoclonal antibody could be used to measure VIII:C bound to polystyrene in an RIA. The antibody shows negligible inactivation of VIII:C. The antibody coupled to agarose, quantitatively removes coagulant from solutions of dissociated VIII:C but will not remove VIII: C activity from plasma (or other sources of undissociated factor VIII complex). The VIII: C absorbed onto the antibody beads is still active. Less than 10% of input activity is retrievable from the beads using chaotropic ions, but 60% of the VIII: C activity is obtained with organic solvent elution. When analyzed by SDS gel electrophoresis, the active protein is indistinguishable from that eluted in inactive form, at low pH, from human inhibitor columns.
The porcine VIII: C activity isolated using the antibody shows a 2 to 3 ± 105fold purification based on units. VIII: c per A280.
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