Production and characterization of a monoclonal antibody against the beta-adrenergic agonist ractopamine

A monoclonal antibody was generated toward the beta-adrenergic agonist ract opamine hydrochloride {(1R*,3R*), (1R*,3S*)-4-hydroxy-alpha-[[[3-(4-hydroxy phenyl)-1-methylpropyl]amino]methyl]benzene-methanol hydrochloride}. Ractop amine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype Ig G1 kappa, was used for the development of an immunoassay. The selected anti body was specific for racemic ractopamine with an IC50 of 2.69 +/- 0.36 ng/ mL (n = 15). Antibody binding toward ractopamine was stereoselective with ( 1R,3R)-ractopamine having an IC50 of 0.55 +/- 0.09 ng/mL (n = 3). IC50 valu es for the (1S,3R)-, (1S,3S)-, and (1R,3S)-ractopamine stereoisomers were 2 .00 +/- 0.37, 140 +/- 23, and 291 +/- 32 ng/mL (n = 3), respectively. Phene thanolamine beta-agonists showed low cross-reactivity. Studies using a seri es of ractopamine metabolites and ractopamine analogues demonstrated struct ural requirements for the antibody binding. A free phenolic group on the N- butylphenol moiety was required for high-affinity binding because methoxyla ted analogues and metabolites glucuronidated at this phenol generally had I C50 values greater than 200 ng/mL. Ractopamine analogues methoxylated or gl ucuronidated at the ethanolamine phenol had IC50 values of 0.7-2.6 ng/mL. L ack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-ph enylalkyl group. In conclusion, a highly specific monoclonal antibody to ra ctopamine hydrochloride was developed that could be of potential utility in screening assays.