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      • Record: found
      • Abstract: found
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      Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library

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          Abstract

          Background

          Nanobodies (Nbs) are single-domain antigen-binding fragments derived from the camelids heavy-chain only antibodies (HCAbs). Their unique advantageous properties make Nbs highly attractive in various applications. The general approach to obtain Nbs is to isolate them from immune libraries by phage display technology. However, it is unfeasible when the antigens are toxic, lethal, transmissible or of low immunogenicity. Naïve libraries could be an alternative way to solve the above problems.

          Results

          We constructed a large camel naïve phage display Nanobody (Nb) library with great diversity. The generated library contains to 6.86 × 10 11 clones and to our best of knowledge, this is the biggest naïve phage display Nb library. Then Nbs against human procalcitonin (PCT) were isolated from this library. These Nbs showed comparable affinity and antigen-binding thermostability at 37°C and 60°C compared to the PCT Nbs from an immune phage-displayed library. Furthermore, two PCT Nbs that recognize unique epitopes on PCT have been successfully applied to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to detect PCT, which showed a linear working range from 10-1000 ng/mL of PCT.

          Conclusion

          We have constructed a large and diverse naïve phage display Nb library, which potentially functioning as a good resource for selecting antigen-binders with high quality. Moreover, functional Nbs against PCT were successfully characterized and applied, providing great values on medical application.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12951-015-0091-7) contains supplementary material, which is available to authorized users.

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          Most cited references23

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          A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks.

          A S Greenberg, D. Avila, M. Hughes … (1995)
          Immunoglobulin and T-cell receptor (TCR) molecules are central to the adaptive immune system. Sequence conservation, similarities in domain structure, and usage of similar recombination signal sequences and recombination machinery indicate that there was probably a time during evolution when an ancestral receptor diverged to the modern-day immunoglobulin and TCR. Other molecules that undergo rearrangement have not been described in vertebrates, nor have intermediates been identified that have features of both these gene families. We report here the isolation of a new member of the immunoglobulin superfamily from the nurse shark, Ginglymostoma cirratum, which contains one variable and five constant domains and is found as a dimer in serum.
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            Isolation of high affinity human antibodies directly from large synthetic repertoires.

            Garry T Allison, O. Hartley, Geoffrey Williams … (1994)
            Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.
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              Domain antibodies: proteins for therapy.

              C Herring, Ian T M Tomlinson, B P Woolven … (2003)
              Occurring naturally in "heavy chain" immunoglobulins from camels, and now produced in fully human form, domain antibodies (dAbs) are the smallest known antigen-binding fragments of antibodies, ranging from 11 kDa to 15 kDa. dAbs are the robust variable regions of the heavy and light chains of immunoglobulins (VH and VL respectively). They are highly expressed in microbial cell culture, show favourable biophysical properties including solubility and temperature stability, and are well suited to selection and affinity maturation by in vitro selection systems such as phage display. dAbs are bioactive as monomers and, owing to their small size and inherent stability, can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities.
              Article 0 comments Cited 88 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Junrong Yan: seuyjr@163.com
                Pingyan Wang: yami_seu@163.com
                Min Zhu: zhumin555111@163.com
                Guanghui Li: ahmaslgh@126.com
                Ema Romão: ema.romao@gmail.com
                Sheng Xiong: xiongsheng@jnu.edu.cn
                Yakun Wan: ywansystemsbiology@gmail.com
                Journal
                Journal ID (nlm-ta): J Nanobiotechnology
                Journal ID (iso-abbrev): J Nanobiotechnology
                Title: Journal of Nanobiotechnology
                Publisher: BioMed Central (London )
                ISSN (Electronic): 1477-3155
                Publication date (Electronic): 6 May 2015
                Publication date PMC-release: 6 May 2015
                Publication date Collection: 2015
                Volume: 13
                Electronic Location Identifier: 33
                Affiliations
                [ ]The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096 PR China
                [ ]Jiangsu Nanobody Engineering and Research Center, Nantong, 226010 PR China
                [ ]Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510630 PR China
                [ ]Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Faculty of Science, Pleinlaan 2, 1050, Brussels, Belgium
                Article
                Publisher ID: 91
                DOI: 10.1186/s12951-015-0091-7
                PMC ID: 4475299
                PubMed ID: 25944262
                SO-VID: f153db73-90d6-48c8-bdb5-4e9173463eea
                Copyright © © Yan et al. 2015
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 13 January 2015
                Date accepted : 15 April 2015
                Categories
                Subject: Research
                Custom metadata
                issue-copyright-statement © The Author(s) 2015

                ScienceOpen disciplines: Biotechnology
                Keywords: bactrian camel,vhh,naïve phage-displayed library,human procalcitonin,biotin-streptavidin-system,sandwich elisa
                Data availability:
                ScienceOpen disciplines: Biotechnology
                Keywords: bactrian camel, vhh, naïve phage-displayed library, human procalcitonin, biotin-streptavidin-system, sandwich elisa

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