ArticleCurrent protocols2026
High-Throughput Isolation of Nanomouse-Derived VHH Domains: A Practical Guide from Immunization to Nanobody Expression.
Article in Current protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
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Who cites it
1 citing paper in PubMed.
- High-Throughput Isolation of Nanomouse-Derived VHH Domains: A Practical Guide from Immunization to Nanobody Expression.Current protocols · 2026Article
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7 authors.
Funding
Abstract
Nanobodies are small but specific heavy chain-only antibody fragments. Their small size, relative stability, and ability to access difficult to reach deep-tissue antigens makes them valuable research, diagnostic, and therapeutic tools. Nanobodies are derived from the variable heavy (VH) domain of heavy chain-only antibodies that are unique to camelids, including alpacas, llamas, and camels. The approaches employed to produce nanobodies, have been evolving and expanding since the initial discovery of heavy chain-only antibodies 30 years ago. Traditional nanobody development involves camelid immunization with a soluble, purified protein, followed by blood collection and processing, enrichment for potent nanobody sequences, and eventual expression and purification of candidate nanobodies for testing and validation. Alternative nanobody generation strategies aim to identify novel nanobodies utilizing synthetic or animal-derived naïve nanobody libraries in combination with phage-, yeast surface-, or ribosome displays for nanobody selection. This article outlines a novel protocol series for nanobody production using a commercially available transgenic "nanomouse", engineered to produce heavy chain-only antibodies containing camelid VH domains from alpacas, dromedaries, and Bactrian camels. These protocols will cover the following aspects: (1) Nanomouse breeding, genotyping, and colony establishment; (2) Nanomouse immunization and tissue collection; (3) RNA extraction from nanomouse immune cells isolated from blood and tissues; (4) Generation and amplification of VHH DNA from nanomouse cDNA; (5) Digestion of VHH DNA and ligation into a phagemid expression vector; (6) Preparation of a screenable Escherichia coli TG1-based phagemid library; (7) Antigen-driven VHH selection using phage display; (8) Single colony VHH ELISA screening; (9) Sequencing of ELISA hits and candidate VHH sequence identification; (10) Geneblock design of candidate VHH and Gibson Assembly into a nanobody expression vector; and (11) Nanobody over-expression and purification. We provide a comprehensive toolkit to facilitate nanobody development and make it more accessible to the greater research community. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Nanomouse breeding, genotyping, and colony establishment Basic Protocol 2: Nanomouse immunization and tissue collection Basic Protocol 3: RNA extraction from nanomouse immune cells isolated from blood and tissues Basic Protocol 4: Generation and amplification of VHH DNA from nanomouse cDNA Basic Protocol 5: Digestion of VHH DNA and ligation into a phagemid expression vector Basic Protocol 6: Preparation of a screenable E. coli TG1-based phagemid library Basic Protocol 7: Antigen-driven VHH selection using phage display Basic Protocol 8: Single colony VHH enzyme-linked immunosorbent assay (ELISA) screening Basic Protocol 9: Sequencing of ELISA hits and candidate VHH sequence identification Basic Protocol 10: Geneblock design of candidate VHH and Gibson Assembly into a nanobody expression vector Basic Protocol 11: Nanobody over-expression and purification.
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