Premium
Sephadex‐based cell‐affinity adsorbents: preparation and performance
Author(s) -
Besselink Geert,
De Korte Dirk
Publication year - 2002
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20010062
Subject(s) - sephadex , antiserum , chromatography , incubation , chemistry , sepharose , glycine , adsorption , biochemistry , antibody , amino acid , biology , enzyme , organic chemistry , immunology
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, ≈300 μ 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 5×10 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 μ l/ml of gel. Compared with this, SpA–Sepharose CL 4B had to be incubated with much larger amounts of antiserum (100–700 μ 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.