| Autor: |
Litwiller, RD, Jenny, RJ, Katzmann, JA, Miller, RS, Mann, KG |
| Zdroj: |
Blood; June 1986, Vol. 67 Issue: 6 p1583-1590, 8p |
| Abstrakt: |
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. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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