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Isolation of phage-antibodies against Eimeria species that infect chickens

Angani, Mary T.; Owen, Jonathan P.; Maddison, Ben C.; Gough, Kevin C.

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Authors

Mary T. Angani

Jonathan P. Owen

Ben C. Maddison

KEVIN GOUGH KEVIN.GOUGH@NOTTINGHAM.AC.UK
Professor of Biochemistry and Pathology



Abstract

Eimeria is one of the most economically important pathogens in poultry production. Diagnosis of infection has the potential to inform treatment and prevention strategies. Here, phage display technology was used to isolate single chain antibodies (scFvs) that had a broad specificity against oocysts from the seven pathogenic species of Eimeria found in poultry. Three such scFvs, representing 2 scFv HCDR3 motifs, were isolated by random picks of clones isolated after five rounds of iterative enrichment (panning) of phage against the seven Eimeria species. Phage-antibody binding to Eimeria oocysts was also interrogated using next generation sequencing of the HCDR3 region of scFv genes contained with phage particles. This analysis demonstrated that the most abundant scFv found after 5 rounds of panning accounted for over >90 % of scFvs. Furthermore, the three scFvs isolated from random picks of clones were the only antibodies that were enriched through each round of panning. They were also seen to be enriched through the stages of phage panning that included binding to the Eimeria oocysts (selection phase) and to be selected against during the stages that consisted solely of phage propagation (growth only phase). The NGS data was further analysed to identify an additional scFv that demonstrated specific enrichment against 3 Eimeria species at the third round of panning and had the same pattern of enrichment during the selection and growth phases of panning. Rescue and analysis of this phage-scFv demonstrated a binder with broad specificity for Eimeria species. The four antibodies with broad specificity detected all seven Eimeria species in immunoassays. The binding of one such scFv that recognised all species was further validated by fluorescent microscopy.

Citation

Angani, M. T., Owen, J. P., Maddison, B. C., & Gough, K. C. (2024). Isolation of phage-antibodies against Eimeria species that infect chickens. Journal of Immunological Methods, 534, Article 113759. https://doi.org/10.1016/j.jim.2024.113759

Journal Article Type Article
Acceptance Date Sep 23, 2024
Online Publication Date Sep 28, 2024
Publication Date 2024-11
Deposit Date Oct 1, 2024
Publicly Available Date Oct 1, 2024
Print ISSN 0022-1759
Electronic ISSN 1872-7905
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 534
Article Number 113759
DOI https://doi.org/10.1016/j.jim.2024.113759
Public URL https://nottingham-repository.worktribe.com/output/40269942
Publisher URL https://www.sciencedirect.com/science/article/pii/S0022175924001443?via%3Dihub

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