Affinity purification of immunoglobulins from chicken egg yolk using a newsynthetic ligand

Due to the peculiar composition of the egg yolk and the lack of specific af finity ligands, Y immunoglobulins are normally purified using complex and t ime consuming procedures involving a combination of precipitation and chrom atographic steps first to extract and capture and then to polish IgY. In th is study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screen ing of combinatorial libraries, and already characterized for its capabilit y to purify immunoglobulins of class G, M, E and A. Soluble proteins were s eparated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the lig and on the commercially available support Emphaze(TM). In a single chromato graphic step TG19318 affinity columns led to an efficient capture of IgY di rectly from crude samples, and with a purity degree higher than 90%, as det ermined by densitometric scanning of SDS-PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4X excess IgY to 1 mi bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per mi of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivat ized matrix was found to be very stable, in terms of ligand leakage and mai ntenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage. (C) 2000 Elsevier Science B.V. All rights reserved.