Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications

Abstract

BackgroundNanobodies (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.MethodsWe 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.ResultsA large and diverse synthetic phage display Nb library with CDR3 regions randomized by trinucleotide cassettes was constructed. The library size was 1.65 × 109 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.ConclusionThis 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.

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

“…Synthetic camelid VHH libraries have been constructed based on natural VHH frameworks. Antigen binding diversity has been introduced by randomization of the CDRs, mainly CDR3 . Interestingly, a semi‐synthetic library has been constructed from 3 unimmunized llamas.…”

Structural and genetic diversity in antibody repertoires from diverse species

“…Monegal et al, 2009 constructed a large naïve llama phage display library that enabled identification of constituents with high affinity binding to FGFR1 [60]. In a related approach, a synthetic phage display nanobody library was built based on a conserved camel single-domain antibody fragment (V HH ) framework and diversity was introduced into the CDR H3 by randomization using synthetic oligonucleotides [61]. While immunization and phage display approaches have met with success in identifying specific binders, Eschrichia coli display with camel V HH 's has also resulted in the identification of high affinity binders [62].…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…Since the entire antigen-binding fragment of HCAb consists of one domain, encoded by a gene fragment of only~360 bp that is easily amplified by PCR, small libraries of~10 6 e10 7 individual transformants are already representative of the immune repertoire [46]. Naive libraries are generated in the same manner from the blood of a non-immunised animal, while synthetic libraries are created, for example, by randomly mutating the amino acids belonging to CDR3 of a V H H exhibiting a robust scaffold [55,56]. Some residues of the CDR3, that are essential to maintain its conformation, must be conserved to ensure the proper folding of the mutant V H Hs [34].…”