Preparation of Ractopamine Single-Chain Variable Fragment and Development of icELISA Based on Immunomagnetic Beads

Abstract

Ractopamine (RAC) is a feed additive that regulates protein synthesis. In this study, an indirect enzyme-linked immunosorbent assay (icELISA) for the detection of RAC residues in the porcine liver was developed based on our specific immunomagnetic bead (IMB) coated with ractopamine-single chain variable fragment (RAC-scFv). The intact RAC-scFv was constructed, and its key amino acid sites were analyzed. Finally, an icELISA based on immunomagnetic beads separation techniques (IMBs) of RAC from the porcine liver was established with an IC50 of 9.15 ng/mL and a linear range from 4.8 to 17.4 ng / mL. The IC50 of the prepared RAC-scFv was 8.7 ng/mL with a linear range of 3.8 to 19.6 ng/mL. The limit of detection (LOD) in the porcine liver of RAC was 4.57 μg/kg, and the recoveries ranged from 72.65 to 89.41% (CV < 15%).

Nafion/Silver Nanoparticles as an Electrochemically Sensitive Interface for the Detection of Ractopamine in Pork Liver

“…Currently, analytical methods for detecting Rac in pork are limited to techniques requiring highly sophisticated sample preparation or costly instrumentation, such as enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), and liquid chromatography–tandem mass spectrometry (LC–MS/MS). − ELISA offers a rapid and portable method for detecting Rac; however, its major drawback is its requirements of complicated time-consuming sample pretreatment and preparation because of the complex matrix of animal tissues . Among instrumental techniques, HPLC enables multicomponent and highly sensitive analysis; however, the HPLC instrument is costly and nonportable, which prevents its widespread application .…”