Preparation and Identification of anti-Ractopamine Monoclonal antibodies

Abstract

This study aimed to prepare high-sensitivity monoclonal antibodies against ractopamine (Rac), which provided a solid foundation for icELISA kit. Mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1,4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma cell lines named R1-B5, R2-B3, R2-C6, and R4C8 were screened out, their corresponding mAbs were of the IgG 1 isotype with k light chain, and the Kafs of all mAbs were between 2.7×10 9 and 4.8×10 9 L/mol. Based on the R1-B5, a heterologous icELISA method was developed, and the working range was from 0.013 to 33.7 ng/mL, with LOD and IC 50 value of 0.007 and 0.67 ng/mL, respectively. Except for a high cross-reactivity (42.7%) to dobutamine, negligible cross-reactivity to other compounds tested was observed. The recoveries of Rac were in the range of 98.2-109.5%, 85.7-110.5% and 97.4-101.8% for cattle muscle, liver and kidney, respectively, with coefficients of variation (CV) values all <10%. When applied in real samples, the correlation coefficient (R 2 ) of ELISA and LC-MS was 0.9373 in cattle muscle. Therefore, this assay can be used for detecting Rac residue in animal products.