Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.

YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)(4) was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate-protein interaction.