Production of monoclonal antibodies for ractopamine residue detection in pork

Ractopamine (RAC) is used as a feed additive to deposit fat and improve the growth rate of food-producing animals. Because RAC residues in meat are unhealthy for consumers, RAC has been banned in many countries and its routine screening is now essential. We aim to generate highly sensitive and specific anti-RAC monoclonal antibodies (mAb) for detecting RAC residues in pork. By means of the hybridoma technique, BALB/c mice were immunised with RAC conjugated with bovine serum albumin. Indirect and competitive enzyme-linked immunosorbent assay (ELISA) identified five hybridoma clones that secrete anti-RAC antibodies. The mAb 10A4 which shows the highest sensitivity was used in an indirect competitive ELISA in a biotin-streptavidin system, which demonstrates 50% inhibition at 0.3 ng/mL and shows detection and quantitation limits at 0.07 ng/mL and 0.13 ng/mL respectively. To evaluate this method, the pork was fortified with RAC at different concentrations. The recovery and coefficient of variation ranges in the intra-variation assay were 91-97% and 3.2-6.7% respectively, whereas those in the inter-variation assay were 90-99% and 6.4-9.9% respectively. The quantification based on the linear range of RAC standard was 0.05-5.0 ng/mL. The anti-RAC mAb generated could be potentially useful for RAC residue detection in pork using the indirect competitive ELISA with the biotin-streptavidin system.