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Related Experiment Videos

Sephadex-based cell-affinity adsorbents: preparation and performance.

Geert Besselink1, Dirk de Korte

  • 1Department of Transfusion Technology, CLB, Sanquin Blood Supply Foundation, P.O. Box 9190, 1066 AC Amsterdam, The Netherlands.

Biotechnology and Applied Biochemistry
|February 9, 2002
PubMed
Summary
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Researchers developed a new method for creating cell-affinity media using Sephadex, Staphylococcus Protein A (SpA), and antibodies. This technique requires less SpA and antiserum, offering an efficient way to bind cells like human red blood cells.

Area of Science:

  • Bioconjugation Chemistry
  • Affinity Chromatography
  • Biomaterial Science

Background:

  • Affinity chromatography is crucial for isolating biomolecules.
  • Developing efficient and cost-effective affinity media is an ongoing challenge.
  • Immobilization of ligands like Protein A and antibodies onto solid supports is key.

Purpose of the Study:

  • To derivatize Sephadex with Staphylococcus Protein A (SpA) and cell-specific antibodies.
  • To evaluate the cell-binding capacity of the resulting affinity material.
  • To compare this novel approach with commercially available SpA-Sepharose.

Main Methods:

  • Carboxylation of Sephadex G-10 with glycine, followed by activation with NHS-carbodiimide.
  • Coupling of SpA to activated Sephadex under specific pH and ionic strength conditions.

Related Experiment Videos

  • Immobilization of cell-specific antibodies onto SpA-derivatized Sephadex and subsequent cell binding assays.

    • Main Results:

    • Complete SpA coupling to Sephadex was achieved at pH 4.0, with a capacity of ~300 µg protein/ml gel.
    • The SpA-glycine-Sephadex G-10 successfully bound up to 5 x 10^8 human red blood cells/ml gel.
    • Significantly lower amounts of SpA and antiserum were required compared to SpA-Sepharose CL 4B.

    Conclusions:

    • The described method provides an efficient and economical way to prepare cell-affinity media.
    • This approach reduces the need for expensive reagents like SpA and antisera.
    • The developed Sephadex-based affinity material demonstrates high cell-binding capacity.