Sensitive enzyme immunoassay of colistin and its application to detect residual colistin in rainbow trout tissue

Abstract

An antibody against colistin (CL), an antibiotic effective for gram-negative bacteria, was produced in rabbits immunized with a colistin-protein conjugate. The conjugate was prepared by a novel and convenient procedure devised to couple an amino group of CL to thiol groups of bovine serum albumin (BSA) introduced by thiol exchange reduction of its disulfide bonds with dithiothreitol, using N-(m-maleimidobenzoyloxy)succinimide (MBS) as a cross-linker. Enzyme labeling of CL with β-D-galactosidase was performed by utilizing another crosslinker, N-(γ-maleimidobutyryloxy)succinamide, by means of a convenient labeling method. A double antibody enzyme immunoassay of CL, which could determine as little as 30 ng/mL of CL, was developed using labeled CL and anti-CL antiserum. With this assay, drug levels were easily determined in fish tissue after CL administration. The enzyme immunoassay should provide a useful tool for detection and quantitation of residual drugs in food and related products.