title: Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications.
abstract: BACKGROUND
  Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. Although the routine procedure to obtain Nbs is to immunize camels with antigens, it is unavailable to immunize a camel when the antigens are highly toxic, pathogenic or nonimmunogenic. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones.
  
  
  METHODS
  We constructed a large and diverse synthetic phage display Nanobody (Nb) library based on the conserved camel single-domain antibody fragment (VHH) framework of cAbBCII10. Diversity was introduced in the complementarity-determining region 3 (CDR3) by means of randomization of synthetic oligonucleotides. Then human prealbumin (PA) and neutrophil gelatinase-associated lipocalin (NGAL) were used to select specific Nbs from this library. Furthermore, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect PA based on horseradish peroxidase (HRP)-conjugated anti-PA Nb isolated from this study and another biotinylated anti-PA Nb obtained from an immune library, in our previous study.
  
  
  RESULTS
  A large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 10(9) CFU/mL and the correct insertion ratio was nearly 100%. A Nb against human PA and against NGAL was successfully isolated from the synthetic library. The obtained anti-PA Nb was effectively used to develop a sandwich ELISA for PA detection and it demonstrated a working range from 50 to 1000 ng/mL, with a limit of detection (LOD) of 27.1 ng/mL.
  
  
  CONCLUSIONS
  This proposed novel synthetic library was a good source for obtaining some antigen-specific Nbs. This approach could provide crucial support to an immune library and a naïve library in the acquisition of specific Nbs, potentially functioning as a great resource for medical diagnostic applications. In addition, we have successfully developed a novel sandwich ELISA to detect PA, which could provide great assistance for clinical PA detection.
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. ahmaslgh@126.com.
authors.forename: Guanghui
authors.initials: G
authors.lastname: Li
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 210009, PR China. hyh@njtech.edu.cn.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Nanobody Engineering and Research Center, Nantong, 226010, PR China. ywansystemsbiology@163.com.
authors.forename: Weijun
authors.initials: W
authors.lastname: Ou
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; D001435:Bacteriophages / Q000235:genetics*; D022801:Complementarity Determining Regions; D004797:Enzyme-Linked Immunosorbent Assay; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000276:immunology; D061905:Single-Domain Antibodies; D018911:Trinucleotide Repeats
chemical_list: D022801:Complementarity Determining Regions; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies