Sephadex-based cell-affinity adsorbents: preparation and performance.
Sephadex was derivatized consecutively with Staphylococcus Protein A (SpA) and cell-specific antibodies, and the binding of cells to the resulting material was examined. For comparison, cell binding to commercially obtained SpA-Sepharose was determined. Sephadex G-10, carboxylated by reaction with glycine and activated subsequently with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide/N-hydroxysuccinimide (NHS), was allowed to react with SpA. Coupling of SpA to NHS-activated glycine-Sephadex appeared to be complete (immobilization capacity, approximately 300 microg of protein/ml of packed gel) when incubation was carried out at pH 4.0, in buffer of low ionic strength. However, incubation at higher pH values (> or = 6.5) led to poor coupling yields. After incubation with rabbit anti-(human red cell) antiserum, and upon mixing with human red blood cells, SpA-glycine-Sephadex G-10 could bind up to 5 x 10(8) red cells/ml of gel. Cell binding increased when the amount of antiserum, added to SpA-glycine-Sephadex G-10 for preparing the affinity gel, was increased from 0.5 to 5 microl/ml of gel. Compared with this, SpA-Sepharose CL 4B had to be incubated with much larger amounts of antiserum (100-700 microl/ml of gel) in order to obtain cell-affinity adsorbent. One obvious advantage of the approach described here is that relatively small amounts of SpA and antisera are needed for preparing cell-affinity media.
Besselink
Geert
G
Department of Transfusion Technology, CLB, Sanquin Blood Supply Foundation, P.O. Box 9190, 1066 AC Amsterdam, The Netherlands.
de Korte
Dirk
D
D000327:Adsorption; D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification; D002234:Carbodiimides / Q000737:chemistry; D002448:Cell Adhesion; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry* / Q000648:ultrastructure; D004912:Erythrocytes / Q000378:metabolism / Q000648:ultrastructure; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007106:Immune Sera / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry*; D009994:Osmolar Concentration; D011485:Protein Binding; D011817:Rabbits; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000378:metabolism / Q000648:ultrastructure; D013997:Time Factors
D000345:Affinity Labels; D001798:Blood Proteins; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003911:Dextrans; D007106:Immune Sera; D007202:Indicators and Reagents; D013205:Staphylococcal Protein A; D012685:Sepharose; C025614:sephadex; D005998:Glycine