Commercially available anti-human/-mouse monoclonal antibodies cross-reactive to agriculture and wildlife species

Authors: Byrne, Kristen; Loving, Crystal

Submitted to: Conference Research Workers Disease Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 11/12/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Objective: Animal health is a key component of “One Health”, reducing the spread of zoonotic diseases and ensuring a safe food supply. However, reagents required to monitor immune status in agricultural and wildlife species are lacking compared to available human and rodent reagents. Therefore, our objective was to increase access to immune specific reagents by identifying commercially available monoclonal antibodies (mAbs) that cross-reactive with agricultural and wildlife species. Methods: Commercially available flow cytometric panels of over 400 anti-human and anti-mouse mAbs were selected for cross-reactivity testing against seven agriculture and wildlife species. Species include bison, chicken, dairy cows, pigs, sheep, turkeys, or white tail deer and utilized either single cell suspensions from immune tissues (spleen, lymph node, thymus, or bursa of Fabricius) or peripheral blood mononuclear cells (PBMCs). Tissue cell were stained either directly after isolation, while PBMCs were cultured for 16h with or without various stimulants (ConA, ionomycin, LPS, and/or PMA) to increase expression of activation markers. Cells were stained with a fixable viability dye and then manufacturer’s protocol was followed for staining of approximately 5e5 cells per mAb. Data were acquired on a BD FACSymphony, and percent positive determined using a fluorescent minus one gating strategy after exclusion of doublets and dead cells. Results: Of the 417 monoclonal antibodies tested, 230 were punitive cross-reactive (at least 2-fold greater reactivity than the matched isotype control) to at least one species and sample type (PBMC or tissue) combination. Turkeys and chickens had the lowest punitive cross-reactivity with 47 and 79 mAbs identified respectively, while pig PBMCs had the highest reactivity with 115 mAbs identified. Putative cross-reactive mAbs included rare markers such as CD180, a B cell subset marker, with 0.9% positive events on pig PBMCs, to very abundant markers such as MHC Class I with 97% of bovine PBMCs positive. We identified 32 mAbs as cross-reactive with at least 3 species and at least 5% positive events and plan to further validate specificity and cross-reactivity. To date, three of those 32 mAbs, B-ly4 (CD21), 3A6 (CD166), and Tu39 (MHC Class II), have been validated via flow cytometry with directly conjugated mAbs and confirmed as cross reactive in bison, sheep, cattle, and pigs. Conclusions: Identification of commercially available, fluorescently labeled mAbs cross-reactive to livestock and wildlife immune cells will enhance relevant research programs across the globe. While further research is needed to confirm the punitive cross-reactive clones identified herein, this research may be useful to other agriculture and wildlife researchers as a framework to quickly identify commercially available cross-reactive mAbs and expand research capabilities.