title: Characterization and applications of Nanobodies against human procalcitonin selected from a novel naïve Nanobody phage display library.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, PR China. seuyjr@163.com.
authors.forename: Junrong
authors.initials: J
authors.lastname: Yan
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, PR China. yami_seu@163.com.
authors.forename: Pingyan
authors.initials: P
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, PR China. zhumin555111@163.com.
authors.forename: Min
authors.initials: M
authors.lastname: Zhu
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, PR China. ahmaslgh@126.com.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Li
authors.affiliation: Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Faculty of Science, Pleinlaan 2, 1050, Brussels, Belgium. ema.romao@gmail.com.
authors.forename: Ema
authors.initials: E
authors.lastname: Romão
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou, 510630, PR China. xiongsheng@jnu.edu.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Xiong
authors.affiliation: The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, PR China. ywansystemsbiology@gmail.com.|Jiangsu Nanobody Engineering and Research Center, Nantong, 226010, PR China. ywansystemsbiology@gmail.com.
authors.forename: Yakun
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002116:Calcitonin / Q000276:immunology*; D015740:Calcitonin Gene-Related Peptide; D002162:Camelus / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008214:Lymphocytes / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011498:Protein Precursors / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C000606589:CALCA protein, human; D000939:Epitopes; D019151:Peptide Library; D011498:Protein Precursors; D061905:Single-Domain Antibodies; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide