Abstract
Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.
Highlights
Generation of recombinant FMD virus (FMDV) antibodies for advancing diagnostics buffer (1% w/v BSA in PBS containing 0.1% v/v Tween-20 (PBS-T)) at 37 ̊C for 1h, and incubated either with 100μL of monoclonal antibodies (MAbs) D9 or concentrated mouse/rabbit D9 chimera at 37 ̊C for 1h
The antigenic site recognised by MAb D9 and SD6 are both mapped to the GH loop of VP1 but have different lengths and sequences [34, 72, 73]
The Fab/peptide structure shows that, consistent with the length of the complementarity-determining regions (CDRs) and the above characteristics of peptide binding antibodies, the CDRs of SD6 form a Generation of recombinant FMDV antibodies for advancing diagnostics groove that is occupied by the peptide [85]
Summary
Foot-and-mouth disease (FMD) is one of the most prevalent epizootic animal diseases affecting economically important livestock (e.g. cattle, buffalo, sheep, goats and pigs) and numerous. Generation of recombinant FMDV antibodies for advancing diagnostics and the United Kingdom Department for Environment, Food and Rural Affairs, projects SE1127, SE1128 and SE1129 to DPK, https://www. Gov.uk/government/organisations/department-forenvironment-food-rural-affairs, and the Medical Research Council, project MR/N00065X/1 to DIS, https://mrc.ukri.org/ and the Wellcome Trust, project 101122/Z/13/Z to DIS, https://wellcome.ac. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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