Preparation and interaction mechanism analysis of single‑chain fragment variables against phenylethanolamine A

This is the first report on the screening,expression,and recognition mechanism analysis of single-chain fragment variable(scFv)against phenylethanolamine A(PEAA),a newly emergedβ-adrenergic agonist illegally used as a feed additive for growth promotion.The PEAA-specific scFv scFv,called scFv-32,was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E.coli system.The ic-ELISA results revealed an IC_(50)value of 10.34μg/L for scFv-32 and no cross-reactivity with otherβ-adrenergic agonists.Homology modeling and molecular docking revealed the key binding sites VAL178,TYP228,and ASP229.One hydrogen bond,two pisigma bonds,and one pi-pi bond maintain the formation of the antibody‒drug complex.Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity,which confirmed the accuracy of the theoretical analysis.These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.

Electrochemical Determination of Phenylethanolamine A Based on Nafion/MWCNTs/AuNPs Modified Carbon Electrode

Phenylethanolamine A （PEA）, a fl-agonist, was found to be illegally used as a growth promoter in pigs last year, causing Chinese government＇s great attention. Here, a sensitive electrochemical method was developed for detecting PEA by immobilization of gold nanoparticles （AuNPs）, multiwalled carbon nanotubes （MWCNTs） and Nation on the surface of a glassy carbon electrode （GCE）. The Nafion/MWCNTs/AuNPs film was characterized by scanning electronic micrographs （SEM） and electrochemical impedance spectroscopy （EIS）. The electrochemical behaviors of PEA at the modified GCE were investigated in detail. The synergetic effects of AuNPs, MWCNTs and Nation amplify the electrochemical reduction signal of PEA, and result in high sensitivity for PEA determination. Under the optimal conditions, the electrochemical sensor shows a wide linear range of 0.01 to 10 μmol/L with a detection limit of 0.005μmol/L. Moreover, the fabricated sensor presents high selectivity and long-term stability, which paves a new way for simple, rapid, sensitive detection of PEA.