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.
Highlights
Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools
PBS, NaHCO3, H2SO4, NaIO4, NaBH4, NaCl, MgCl2, tryptone, yeast extract, polyethylene glycol (PEG) 6000, D-biotin, tween-20, bovine serum albumin (BSA), ampicillin, kanamycin, imidazole, glycerol, ethylene glycol and glucose were obtained from Sangon Biotech (China). 24-well cell culture plate was purchased from Corning (USA)
Construction of a synthetic phage display Nb library cAbBCII10 has been proved to be an excellent candidate as an universal framework based on its good stability, good expression level and functional property in the absence of the conserved disulfide bond [29]
Summary
Nanobodies (Nbs) have proved their great value as therapeutic molecules and clinical diagnostic tools. A synthetic phage display library is an alternative to generate Nbs against such targets, besides all the other ones. Since the technique of generation monoclonal antibodies has been discovered, it has showed great value as both diagnostic and therapeutic tool. The manufacturing processes in the production capacity of therapeutic antibodies in eukaryotic systems will cause a high cost [1]. The large size of monoclonal antibodies may disturb efficient tissue accessibility and penetration [2]. Such problems could be solved by obtaining smaller antigen-binding antibody fragments [3]
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