Reusability of Avidin-Biotinylated Immunoglobulin Y Columns in Immunoaffinity Chromatography
✍ Scribed by Hyun-Ock Kim; Timothy D. Durance; Eunice C.Y. Li-Chan
- Publisher
- Elsevier Science
- Year
- 1999
- Tongue
- English
- Weight
- 228 KB
- Volume
- 268
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 Co-vaLink 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.