Reusability of avidin-biotinylated immunoglobulin Y columns in immunoaffinity chromatography

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatog raphy was examined by repeated use and regeneration. An enzyme-linked immun osorbent assay-elution assay using CovaLink NH microtiter plates was used t o find the optimal conditions for regeneration of columns. Actigel avidin-b iotinylated IgY column retained about 90% of its initial IgG binding capaci ty after 50 cycles, with 0.1 M glycine-HCl buffer, pH 2.8, as eluent, requi ring no regeneration. However, IgG binding capacity of UltraLink avidin-bio tinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycl es, respectively, with the commercial eluent, Actisep. Results from the Cov alink NH system agreed with those from UltraLink avidin-biotinylated IgY co lumns. The Ultralink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine-HCl, pH 1.6, to dissociate biotinylated IgY antib odies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY t o the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidi n-biotinylated IgY columns are reusable with or without regeneration depend ing on the avidin-immobilized matrix. (C) 1999 Academic Press.