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Biotechnology and Applied Biochemistry (2002) 35, (55–60) (Printed in Great Britain)

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Sephadex-based cell-affinity adsorbents: preparation and performance

Geert Besselink and Dirk de Korte¹

Department of Transfusion Technology, CLB, Sanquin Blood Supply Foundation, P. O. Box 9190, 1066 AC Amsterdam, The Netherlands

Key words: antibody, hydrogel, immobilization, Sepharose, Staphylococcus protein A.

Abbreviations used: SAG-M, 150 mM NaCl/1.25 mM adenine/47 mM glucose/29 mM mannitol; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide; NHS, N-hydroxysuccinimide; SpA, Staphylococcus Protein A.

¹To whom correspondence should be addressed (e-mail D_de_Korte@clb.nl).

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 µg 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 µl/ml of gel. Compared with this, SpA–Sepharose CL 4B had to be incubated with much larger amounts of antiserum (100–700 µl/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.

Received 3 August 2001; accepted 19 November 2001

Portland Press Ltd © 2002