Preparation of anti-chloramphenicol rabbit antibody

Abstract

Chloramphenicol (CAP) is a broad-spectrum antibiotic of high toxicity on human therefore it is being strictly controlled in food. We are interested in the development of a method of effective extraction of CAP in food based on the immunological principle using specific anti-CAP antibody combining with LC/MS/MS for the analysis of the residual CAP in food meeting the international standard. In this article, we reported experimental results on the preparation of anti-CAP rabbit antibody. Conjugative antigen between CAP and the carrier protein, bovine serum albumin, BSA (CAP-BSA) was successfully synthesized from chloramphenicol succinate and BSA with a BSA conjugating efficiency of more than 70 %, and the presence of CAP in the conjugative antigen was confirmed by ELISA method. The CAP-BSA antigen could cause good immune response in rabbit by the first antigen injection and induce the increasing production of anti-CAP antibody in rabbit serum from the third antigen injection which reached maximal value after the fifth injection. Anti-CAP antibody was purified from rabbit anti-serum in two steps: i) Removing of albumin and other non antibody proteins by 35–37 % saturated ammonium sulphate; ii) Elimination of anti-BSA antibody by the Sepharose-BSA specific affinity chromatography column. The ability of the purified anti-CAP antibody to interact and bind with CAP molecules in CAP-spiked sample was proved using a Sepharose-Anti-CAP antibody chromatography column which was made by conjugating the purified anti-CAP antibody with Sepharose beads.