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/Nhydroxysuccinimide (NHS), was allowed to react with SpA. Coupling of SpA to NHS-activated glycine-Sephadex appeared to be complete (immobilization capacity,(almost equal) 300 (mu)g 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 (>= 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 5×108 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 (mu)l/ml of gel. Compared with this, SpA-Sepharose CL 4B had to be incubated with much larger amounts of antiserum (100-700 (mu)l/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.