Improved preparation of a chitosan-based immunoaffinity column using antibody against methandrostenolone as ligand

Abstract

Chitosan is obtained by N-deacetylation of chitin, which is the second most abundant natural biopolymer. In this study, an improved preparation and characterisation of a chitosan-based immunoaffinity chromatography (IAC) column were performed. The immunoaffinity adsorbent was prepared by covalently coupling monoclonal antibody (mAb) against methandrostenolone (MA) to glutaraldehyde cross-linked chitosan (chitosan CL). Scanning electron micrograph of chitosan CL indicated that the shape of the particles was spherical with a diameter range from about 300 to 500 µm. Infrared spectral analysis suggested that the immunoaffinity adsorbent was successfully prepared by coupling antibody with chitosan CL. For chromatographic extraction, 90% methanol was selected as eluant. Under optimum conditions, the maximum binding capacity of the IAC column was 3900 ng MA/mL adsorbent. The average recovery of 50, 250 and 500 ng of MA standards from IAC columns was 96.9% with a relative standard deviation among columns of 1.48%. After eight uses, the extraction recovery of the IAC column remained 82.1%. To further verify the effect of matrices on the extraction efficiency, the IAC columns were challenged with MA-fortified animal tissue and feed samples. Recoveries were 84.9–87.1%, demonstrating the effectiveness of these IAC columns for sample clean-up in MA residue determination.