Abstract
Nanobodies (VHH) from camelid antibody libraries hold great promise as therapeutic agents and components of immunoassay systems. Synthetic antibody libraries that could be designed and generated once and for various applications could yield binders to virtually any targets, even for non-immunogenic or toxic ones, in a short term. One of the most difficult tasks is to obtain antibodies with a high affinity and specificity to polyglycosylated proteins. It requires antibody libraries with extremely high functional diversity and the use of sophisticated selection techniques. Here we report a development of a novel sandwich immunoassay involving a combination of the synthetic library-derived VHH-Fc fusion protein as a capture antibody and the immune single-chain fragment variable (scFv) as a tracer for the detection of pregnancy-associated glycoprotein (PAG) of cattle (Bos taurus). We succeeded in the generation of a number of specific scFv antibodies against PAG from the mouse immune library. Subsequent selection using the immobilized scFv-Fc capture antibody allowed to isolate 1.9 nM VHH binder from the diverse synthetic library without any overlapping with the capture antibody binding site. The prototype sandwich ELISA based on the synthetic VHH and the immune scFv was established. This is the first successful example of the combination of synthetic and immune antibody libraries in a single sandwich immunoassay. Thus, our approach could be used for the express isolation of antibody pairs and the development of sandwich immunoassays for challenging antigens.
Key points
• Heavily glycosylated PAG Bos Taurus were used for immune library construction and specific scFv isolation by phage display.
• Nanomolar affinity VHH for PAG was selected from the original synthetic nanobodies library.
• A novel VHH/scFv-based immunoassay for Bos Taurus pregnancy determination was developed.
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07 July 2022
The sizes of the images of Figs. 1, 4 and 6 are incorrect. This has been updated and corrected to the appropriate size.
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Acknowledgements
This study was financially supported by the State Committee on Science and Technology. We would like to thank Elena Tumar and Elena Kisileva at the Institute of Bioorganic Chemistry of NASB for their kind assistance with mouse immunizations.
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DD, MS, and AG designed the study; IK and ES performed bovine cotyledon extraction and bovine sample preparation; MS purified PAGs; DD performed phage display selections; MS and AlK purified and analyzed anti-PAG VHH and scFv; AnK produced VHH-Fc in FreeStyle™ 293-F cells; DD analyzed the data and wrote the manuscript. All authors read and approved the manuscript.
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The animal experiments were approved by the Institute of Experimental Veterinary Medicine named S. N. Vyshelesski, the Animal Care and Use Committee. The study was performed in accordance with the Belarussian legal and ethical requirements for appropriate animal procedures. Ruminants were not purposely euthanized for this study. Tissue samples were taken during the conventional slaughter process. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
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Dormeshkin, D., Shapira, M., Karputs, A. et al. Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Appl Microbiol Biotechnol 106, 5093–5103 (2022). https://doi.org/10.1007/s00253-022-12022-w
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DOI: https://doi.org/10.1007/s00253-022-12022-w