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RD Litwiller, RJ Jenny, JA Katzmann, RS Miller and KG Mann
Monoclonal antibodies to human protein S have been prepared using
established hybridoma technology. One antibody was isolated that binds
protein S only when Ca2+ is present; others bind antigen equally well in
the presence or absence of EDTA. Other antibodies display a diminished
affinity for protein S in the presence of EDTA. Purified immunoglobulins
from cell lines displaying Ca2+ dependence were immobilized and used to
purify protein S from fractions obtained by barium precipitation of
citrated plasma, ammonium sulfate fractionation, and chromatography on
diethylaminoethanol (DEAE)- Sephadex and dextran sulfate agarose.
Essentially homogeneous protein S was isolated from the
barium-citrate-adsorbed, 35% ammonium-sulfate- soluble proteins using a
totally Ca2+-dependent antibody and EDTA elution. Protein S and several
substances of higher mol wt were bound directly from plasma by a partially
Ca2+-dependent antibody and were eluted partially with EDTA and NaCl and
finally with NaSCN. The largest and most abundant of the high mol wt
materials is likely protein S- complement C4b-binding protein complex. The
immunoaffinity-isolated protein S was found to be indistinguishable from
conventionally isolated protein S in terms of activity, sodium dodecyl
sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) mobility, and by
high- performance liquid chromatography (HPLC). These studies establish
reagents that can probe the structure of protein S and isolate protein S in
its free and complexed forms.
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| Copyright © 1986 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
