Preparation of an immunoaffinity chromatography column for chloramphenicol binding

Abstract

Anti-chloramphenicol rabbit antibody was covalently bound to Sepharose CL 4B-CNBr gel at 5, 10 and 15 mg antibody per mL gel and was fed into columns to prepare three respective immunoaffinity chromatography columns for chloramphenicol (CAP) binding. Examination of CAP-binding efficiency of these columns being fed with 0.5, 1 and 10 ng CAP showed that the column with 10 mg antibody per ml gel (IAC-CAP-10 column) could bind more than 90 % of the CAP amount having been fed into the column at all three examined CAP amounts. This IAC-CAP-10 column showed a coefficient of variants (CV) between columns upon CAP binding of 5.18 % for 1 ng fed CAP and 1.98 % for 10 ng, respectively, a loading capacity of 153,37 ± 10,32 ng CAP, stable CAP-binding performance during at least 2 years of preservation at 4 °C. The coupling of this IAC-CAP-10 column with liquid chromatography - tandem mass spectrometry LC MS/MS could make CAP analysis become possible with a limit of detection LOD of 0.033 ng and a limit of quantification LOQ of  0.101 ng. The column could also bind to the other two phenicol antibiotics: florfenicol and thiamphenicol with a binding efficiency of 90.73 % for florfenicol and 84.29 % for thiamphenicol.