The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9...The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.展开更多
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
The fragile antibody leads to a great challenge as a scaffold to fabricate the luminescent metal nanoclusters using one-pot method.This study presents a stable single-chain anti-body(scFv57R-ATS)for the fabrication of...The fragile antibody leads to a great challenge as a scaffold to fabricate the luminescent metal nanoclusters using one-pot method.This study presents a stable single-chain anti-body(scFv57R-ATS)for the fabrication of luminescent gold nanoclusters(AuNCs@scFv57R-ATS)and a quick,sensitive rabies virus detection in living cells.In this paper,AuNCs@scFv57R-ATS was designed to specifically recognize antigen RV in modified HeLa cells,which promoted the demonstration of metal nanocluster fluorescent probes for antigen targeting and therapy.展开更多
文摘The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.
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.
文摘The fragile antibody leads to a great challenge as a scaffold to fabricate the luminescent metal nanoclusters using one-pot method.This study presents a stable single-chain anti-body(scFv57R-ATS)for the fabrication of luminescent gold nanoclusters(AuNCs@scFv57R-ATS)and a quick,sensitive rabies virus detection in living cells.In this paper,AuNCs@scFv57R-ATS was designed to specifically recognize antigen RV in modified HeLa cells,which promoted the demonstration of metal nanocluster fluorescent probes for antigen targeting and therapy.
