McCreath G E, Chase H A
Department of Chemical Engineering, University of Cambridge, UK.
J Mol Recognit. 1996 Sep-Dec;9(5-6):607-16. doi: 10.1002/(sici)1099-1352(199634/12)9:5/6<607::aid-jmr308>3.0.co;2-s.
Perfluorocarbon affinity emulsions have been generated by immobilizing concanavalin A onto the surface of triazine-activated perfluorocarbon droplets. Immobilized concanavalin A densities of 0.1, 0.7 and 2.1 mg/ml were obtained by varying the concentration of cyanuric chloride used for activation. The affinity emulsions were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of 1 x 10(9) cells, 4.6 x 10(9) and 6 x 10(9) cell/ml, respectively. Optimal conditions for the elution of bound cells were obtained by studying inhibition curves of concanavalin A-mannan precipitation using simple sugars. Methyl alpha-D-mannopyranoside showed the greatest inhibitory power with 50 per cent inhibition displayed at a concentration of 0.05 mM. Experiments carried out examining the concentrations of methyl alpha-D-mannopyranoside necessary to dissociate a concanavalin A-mannan precipitate demonstrated that at least a seven-fold higher concentration of methyl alpha-D-mannopyranoside was required for dissociation than that required for inhibition of its formation. The efficiency of elution of bound yeast cells was found to be dependent on the concentration of methyl alpha-D-mannopyranoside used, the time of elution and the immobilized ligand density. Thus, 100 per cent elution was obtained with a concanavalin A affinity emulsion (0.1 mg/ml) by incubation with 500 mM methyl alpha-D-mannopyranoside for 1 h.
通过将伴刀豆球蛋白A固定在三嗪活化的全氟碳液滴表面，制备了全氟碳亲和乳液。通过改变用于活化的三聚氯氰的浓度，获得了固定化伴刀豆球蛋白A密度为0.1、0.7和2.1mg/ml的乳液。发现亲和乳液能够从溶液中吸附酿酒酵母细胞，吸附容量分别为1×10⁹个细胞/ml、4.6×10⁹个细胞/ml和6×10⁹个细胞/ml。通过研究使用单糖的伴刀豆球蛋白A-甘露聚糖沉淀抑制曲线，获得了洗脱结合细胞的最佳条件。α-D-甲基甘露糖苷显示出最大的抑制能力，在浓度为0.05mM时表现出50%的抑制率。研究解离伴刀豆球蛋白A-甘露聚糖沉淀所需的α-D-甲基甘露糖苷浓度的实验表明，解离所需的α-D-甲基甘露糖苷浓度至少比抑制其形成所需的浓度高7倍。发现结合酵母细胞的洗脱效率取决于所用α-D-甲基甘露糖苷的浓度、洗脱时间和固定化配体密度。因此，通过与500mMα-D-甲基甘露糖苷孵育1小时，使用伴刀豆球蛋白A亲和乳液（0.1mg/ml）可实现100%的洗脱。
