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 ...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.展开更多
Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural ana...Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues,the preparation of highly specific antibodies is crucial.In this study,a novel hapten DEX-GA was designed and synthesized,and the monoclonal antibody(mAb)3D1 was successfully prepared based on the hapten.The mAb 3D1 has the 50%inhibitory concentration of 0.38 ng/mL for dexamethasone,with cross-reactivity below 7.45%against twelve glucocorticoids including betamethasone,prednisone,triamcinolone and beclomethasone.Based on this antibody,indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)and colloidal gold immunochromatography assay(GICA)were developed for accurate detection of dexamethasone in milk and animal tissues.The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215μg/kg,which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method.The GICA method established in this study had a visual limit of detection of 0.3μg/L in milk.Notably,this constitutes the first application of GICA in animal tissues,demonstrating a visual detection limit of 0.2μg/kg.The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.展开更多
基金the National Natural Science Foundation of China(32072920)the Fundamental Research Funds for the Central Universities(2662022DKPY007)the HZAU-AGIS Cooperation Fund(SZYJY2022024).
文摘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.
基金funded by the National Natural Science Founda-tion of China(32573424)the National Key Research and Development Programs of China(2023YFD1301001,2024YFF1105705)the Hubei Provincial Natural Science Foundation(2024AFB146).
文摘Dexamethasone,a long-acting glucocorticoid,exhibits considerable structural similarity to other glucocorticoids.To avoid false-positive results in immunoassays due to cross-reactivity of antibodies with structural analogues,the preparation of highly specific antibodies is crucial.In this study,a novel hapten DEX-GA was designed and synthesized,and the monoclonal antibody(mAb)3D1 was successfully prepared based on the hapten.The mAb 3D1 has the 50%inhibitory concentration of 0.38 ng/mL for dexamethasone,with cross-reactivity below 7.45%against twelve glucocorticoids including betamethasone,prednisone,triamcinolone and beclomethasone.Based on this antibody,indirect competitive enzyme-linked immunosorbent assay(ic-ELISA)and colloidal gold immunochromatography assay(GICA)were developed for accurate detection of dexamethasone in milk and animal tissues.The limits of detection of ic-ELISA in milk and animal tissues ranged from 0.132 to 0.215μg/kg,which was a 1-3-fold increase in sensitivity compared with the previously reported ic-ELISA method.The GICA method established in this study had a visual limit of detection of 0.3μg/L in milk.Notably,this constitutes the first application of GICA in animal tissues,demonstrating a visual detection limit of 0.2μg/kg.The accuracy and reliability of both ic-ELISA and GICA were validated using liquid chromatography-tandem mass spectrometry.
