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Figure 1. Screening and detection of lyszozyme-specific ligands during the consecutive rounds of panning (A) Schematic procedure for the panning of lysozyme specific nanbodies in phage display. (B) The enrichment of phages from each panning. Phages were monitored by ELISA in comparison with the negative control (BSA). The third and fourth rounds of biopanning on immobilized lysozyme antigen (positive well) showed the strongest signal in ELISA. The assay was performed in triplicate and error bar represents the standard deviation. From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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Figure 2. Alignment of NbLx sequences with human VH consensus sequence (hVH) The hypervariable regions are shown in bold. The VHH hallmark amino acids (V42Y/F, G49E, L50R, and W52Xaa) and the Cys residues involved in the intra-domain (C23 and C104) disulfide bond are underlined and highlighted in gray, respectively (IMGT numbering). From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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Figure 3. Alignment of the NbL42 V-REGION protein with the V-REGION of the closest germline V-GENE The third line of the alignment shown in bold indicated the amino acids in NbL42 which are different from those in the human closest germline. From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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Figure 4. Characterization of VHH antibodies (A) The Ni-NTA-affinity purified nanobodies NbL23 (lane 1, 17 kDa), NbL114 (lane 2, 16.5 kDa) and NbL42 (lane 3, kDa) are >95% pure analyzed with SDS-PAGE. (B) Reactivity of nanobodies (10 μg/ml) was tested with indirect ELISA against lysozyme (5 μg/ml) and BSA. An allinase-specific nanobody VHHA4 was used as negative control. (C) Thermal stability of NbL42 was compared with two VHHs and one commercially available mouse monoclonal anti-lysozyme antibody NYRHEL. Antibodies were incubated at increasing temperatures for 30 min, re-equilibrated to room temperature, and then assayed in the standard lysozyme-specific ELISA. The values are the mean of three independent determinations. From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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Figure 5. Inhibition of lysozyme activity by NbL42 (A) Competitive binding between lysozyme specific antibodies and small molecular weight inhibitor. (B) IC<sub>50</sub> determinations for the different inhibitors. (GlcNAc)<sub>3</sub> and enzyme inhibition by CDR3 only VHH antibody NbL42 compared with other lysozyme-specific nanobodies and an unrelated nanobody VHHA4. The residual hydrolytic activity of lysozyme in the presence of various concentrations of inhibitors is plotted relative to that measured in their absence. From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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Figure 6. The lysozyme-binding and enzyme-inhibiting activities of different NbL42 variants (A) Indirect ELISA detection of NbL42 and its three variants (10 μg/ml) binding with lysozyme (5 μg/ml). (B) Inhibition of lysozyme activity by NbL42 and its variants. The residual hydrolytic activity of lysozyme in the presence of various concentrations of inhibitors was determined. From: A camel anti-lysozyme CDR3 only domain antibody selected from phage display VHH library acts as potent lysozyme inhibitor Acta Biochim Biophys Sin (Shanghai). 2017;49(6): doi: /abbs/gmx037 Acta Biochim Biophys Sin (Shanghai) | © The Author Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences. All rights reserved. For permissions, please
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