Reusability of Avidin-Biotinylated Immunoglobulin Y Columns in Immunoaffinity Chromatography

• Published in: Analytical biochemistry 1999-03, Vol.268 (2), p.383-397
• Main Authors: Kim, Hyun-Ock, Durance, Timothy D., Li-Chan, Eunice C.Y.
• Format: Article
• Language: English
• Subjects: Animals; Avidin; avidin-biotinylated IgY; Biotinylation; Cattle; Chromatography, Affinity - methods; Enzyme-Linked Immunosorbent Assay; Equipment Reuse; Evaluation Studies as Topic; Guanidine; IgG binding capacity; Immunoglobulin G - isolation & purification; Immunoglobulins; Rabbits; regeneration
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1998.3081

Reusability of avidin-biotinylated IgY columns for immunoaffinity chromatography was examined by repeated use and regeneration. An enzyme-linked immunosorbent assay-elution assay using CovaLink NH microtiter plates was used to find the optimal conditions for regeneration of columns. Actigel avidin-biotinylated IgY column retained about 90% of its initial IgG binding capacity after 50 cycles, with 0.1 M glycine–HCl buffer, pH 2.8, as eluent, requiring no regeneration. However, IgG binding capacity of UltraLink avidin-biotinylated IgY column gradually decreased to 75 and 65% after 10 and 20 cycles, respectively, with the commercial eluent, Actisep. Results from the CovaLink NH system agreed with those from UltraLink avidin-biotinylated IgY columns. The UltraLink avidin-biotinylated IgY column was regenerated twice, by applying 8 M guanidine–HCl, pH 1.6, to dissociate biotinylated IgY antibodies from the column. About 40 and 25% of IgG binding capacities remained after the first and second regeneration. By applying new biotinylated IgY to the treated columns, about 95 and 90% of the initial IgG binding capacity before any treatment were recovered. These results demonstrated that avidin-biotinylated IgY columns are reusable with or without regeneration depending on the avidin-immobilized matrix.