New haptens synthesis, antibody production and comparative molecular field analysis for tetracyclines

In this work, two novel haptens of tetracycline (TC) were synthesized and used to produce a polyclonal antibody for TC. A selective heterologous enzyme-linked immunosorbent assay (ELISA) using the novel antibody was optimized to exhibit an IC50 value as low as 0.7 ng mL(-1) in buffer. The assay was sufficiently sensitive for the analysis of the maximum residue limit (MRL) of TC in milk (100 mg L-1). The analysis of TC in fortified milk samples indicated average recoveries of 84.9-111.8% with inter-assay CV values of 9.34-14.5% after 100-fold buffer dilution without any clean-up step or organic-solvent usage. The cross-reactivities of 17 other TC analogs were tested using the optimized ELISA, and a comparative molecular field analysis (CoMFA) was performed at the 3D level in a quantitative manner to explain the observed cross-reactivities. The results indicated that the CoMFA was a useful tool for understanding the recognition profiles of antibodies in competitive immunoassays for TCs.