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

Abstract

A monoclonal antibody was generated toward the beta-adrenergic agonist ractopamine hydrochloride ¿(1R,3R),(1R, 3S)-4-hydroxy-alpha-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methy l]benzenemethanol hydrochloride¿. Ractopamine-glutarate-keyhole limpet hemocyanin (KLH) was used as the antigen for antibody generation in mice. Clone 5G10, secreted antibody with isotype IgG1kappa, was used for the development of an immunoassay. The selected antibody was specific for racemic ractopamine with an IC(50) of 2.69 +/- 0.36 ng/mL (n = 15). Antibody binding toward ractopamine was stereoselective with (1R,3R)-ractopamine having an IC(50) of 0.55 +/- 0.09 ng/mL (n = 3). IC(50) 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 beta-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 IC(50) values greater than 200 ng/mL. Ractopamine analogues methoxylated or glucuronidated at the ethanolamine phenol had IC(50) 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.

Preparation and Characterization of Olaquindox Polyclonal Antibody

“…During the experimental process, the choice of the suitable carriers, particularly the immunogen carrier was very important. [29][30][31][32] The physico-chemical property of BSA is stable, not changing, and facilitating the chemical treatments, and it can keep high solubility at different pH and ionic strength. 33 As a carrier protein, the pro-source relations between BSA and OVA is distant, with a low level of cross-reactivity, OVA as the carrier protein was chosen, so it was easy to remove the antibodies to the carrier protein BSA, and greatly reduce the labor intensity of the test.…”

Synthesis of Cinnamic Acid Derivatives

“…In a number of studies, it has been found that some viral transfer gene expression products have tyrosine kinase (PTK) activity [1]. Therefore, PTK has become an important breakthrough in studying the mechanism of oncogenes and exploring the treatment of tumor diseases.…”

New haptens and antibodies for ractopamine

“…The structure of hapten partly determines the affinity and specificity of antibody, then performance of immunoassay. Up to now, few attempt has been made to prepare hapten of RAC in the literatures (Haasnoot et al, 1994;Shelver, Smith, & Berry, 2000). The aim of the study was to synthesize new RAC haptens in order to produce polyclonal antibody with high sensitivity and specificity.…”