--- _id: '11462' abstract: - lang: eng text: 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. acknowledgement: 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. article_processing_charge: No article_type: original author: - first_name: Dmitri full_name: Dormeshkin, Dmitri last_name: Dormeshkin - first_name: Michail full_name: Shapira, Michail last_name: Shapira - first_name: Alena full_name: Karputs, Alena last_name: Karputs - first_name: Anton full_name: Kavaleuski, Anton id: 62304f89-eb97-11eb-a6c2-8903dd183976 last_name: Kavaleuski orcid: 0000-0003-2091-526X - first_name: Ivan full_name: Kuzminski, Ivan last_name: Kuzminski - first_name: Elena full_name: Stepanova, Elena last_name: Stepanova - first_name: Andrei full_name: Gilep, Andrei last_name: Gilep citation: ama: Dormeshkin D, Shapira M, Karputs A, et al. Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. 2022;106:5093-5103. doi:10.1007/s00253-022-12022-w apa: Dormeshkin, D., Shapira, M., Karputs, A., Kavaleuski, A., Kuzminski, I., Stepanova, E., & Gilep, A. (2022). Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. Springer Nature. https://doi.org/10.1007/s00253-022-12022-w chicago: Dormeshkin, Dmitri, Michail Shapira, Alena Karputs, Anton Kavaleuski, Ivan Kuzminski, Elena Stepanova, and Andrei Gilep. “Combining of Synthetic VHH and Immune ScFv Libraries for Pregnancy-Associated Glycoproteins ELISA Development.” Applied Microbiology and Biotechnology. Springer Nature, 2022. https://doi.org/10.1007/s00253-022-12022-w. ieee: D. Dormeshkin et al., “Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development,” Applied Microbiology and Biotechnology, vol. 106. Springer Nature, pp. 5093–5103, 2022. ista: Dormeshkin D, Shapira M, Karputs A, Kavaleuski A, Kuzminski I, Stepanova E, Gilep A. 2022. Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development. Applied Microbiology and Biotechnology. 106, 5093–5103. mla: Dormeshkin, Dmitri, et al. “Combining of Synthetic VHH and Immune ScFv Libraries for Pregnancy-Associated Glycoproteins ELISA Development.” Applied Microbiology and Biotechnology, vol. 106, Springer Nature, 2022, pp. 5093–103, doi:10.1007/s00253-022-12022-w. short: D. Dormeshkin, M. Shapira, A. Karputs, A. Kavaleuski, I. Kuzminski, E. Stepanova, A. Gilep, Applied Microbiology and Biotechnology 106 (2022) 5093–5103. date_created: 2022-06-26T22:01:34Z date_published: 2022-08-01T00:00:00Z date_updated: 2023-10-10T07:15:02Z day: '01' department: - _id: GradSch - _id: LeSa doi: 10.1007/s00253-022-12022-w external_id: isi: - '000813677500001' pmid: - '35723693' intvolume: ' 106' isi: 1 language: - iso: eng month: '08' oa_version: None page: 5093-5103 pmid: 1 publication: Applied Microbiology and Biotechnology publication_identifier: eissn: - 1432-0614 issn: - 0175-7598 publication_status: published publisher: Springer Nature quality_controlled: '1' scopus_import: '1' status: public title: Combining of synthetic VHH and immune scFv libraries for pregnancy-associated glycoproteins ELISA development type: journal_article user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87 volume: 106 year: '2022' ...