Production and Characterization of a Monoclonal Antibody against the β-Adrenergic Agonist Ractopamine

Abstract

A monoclonal antibody was generated toward the β-adrenergic agonist ractopamine hydrochloride {(1R*,3R*),(1R*,3S*)-4-hydroxy-α-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methyl]benzenemethanol hydrochloride}. Ractopamine−glutarate−keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1κ, was used for the development of an immunoassay. The selected antibody 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 values 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. Phenethanolamine β-agonists showed low cross-reactivity. Studies using a series of ractopamine metabolites and ractopamine analogues demonstrated structural requirements for the antibody binding. A free phenolic group on the N-butylphenol moiety was required for high-affinity binding because methoxylated analogues and metabolites glucuronidated at this phenol generally had IC50 values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC50 values of 0.7−2.6 ng/mL. Lack of a benzylic hydroxyl group was of less importance to antibody binding than was the correct stereochemical orientation (3R) of ractopamine's N-phenylalkyl group. In conclusion, a highly specific monoclonal antibody to ractopamine hydrochloride was developed that could be of potential utility in screening assays. Keywords: β-Agonist; ELISA; monoclonal antibody; ractopamine; feed additive; residue