Summary
Four mouse hybridomas secreting monoclonal antibodies specific for human protein S
(PS) have been generated. The antibodies, all of the IgG1 subclass, were designated
S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal
antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited
in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient
on warfarin therapy, and plasma from a patient with no free PS and only PS bound to
C4b-binding protein. The slopes of the curves show that the monoclonal antibodies
reacted equally with all the tested forms of PS indicating that the antigenic site(s)
to which the monoclonal antibodies are directed are present and exposed in free and
bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent
antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed,
ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent
with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally
purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained
by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein
peak containing the bulk of PS was subsequently applied to the immunoadsorbent and
eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify
the isolation of PS from human plasma.
Keywords
Protein S - Calcium-free conformation - Monoclonal antibodies
