Development and Characterization of New Monoclonal Antibodies Against Porcine Interleukin-17A and Interferon-Gamma

Abstract

Current research efforts require a broad range of immune reagents, but those available for pigs are limited. The goal of this study was to generate priority immune reagents for pigs and pipeline them for marketing. Our efforts were aimed at the expression of soluble swine cytokines and the production of panels of monoclonal antibodies (mAbs) to these proteins. Swine interleukin-17A (IL-17A) and Interferon-gamma (IFNγ) recombinant proteins were produced using yeast expression and used for monoclonal antibody (mAb) production resulting in panels of mAbs. We screened each mAb for cross-species reactivity with orthologs of IL-17A or IFNγ and checked each mAb for inhibition by other related mAbs, to assign mAb antigenic determinants. For porcine IL-17A, the characterization of a panel of 10 mAbs identified eight different antigenic determinants; interestingly, most of the mAbs cross-reacted with the dolphin recombinant ortholog. Likewise, the characterization of a panel of nine anti-PoIFNγ mAbs identified four different determinants; most of the mAbs cross-reacted with dolphin, bovine, and caprine recombinant orthologs. There was a unique reaction of one anti-PoIFNγ mAb that cross-reacted with the zebrafish recombinant ortholog. The αIL-17A mAbs were used to develop a quantitative sandwich ELISA detecting the yeast expressed protein as well as native IL-17A in stimulated peripheral blood mononuclear cell (PBMC) supernatants. Our analyses showed that phorbol myristate acetate/ionomycin stimulation of PBMC induced significant expression of IL-17A by CD3+ T cells as detected by several of our mAbs. These new mAbs expand opportunities for immunology research in swine.

Keywords: cytokines; immunoassay; interferon-gamma; interleukin-17A; monoclonal antibodies; swine.

Figure 1

Cross-species binding of αPoIL-17A mAbs. A panel of biotin-labeled αPoIL-17A mAbs were tested for their ability to bind to purified orthologous rIL-17A proteins from species as described in M&M. Shown are mean ODs of duplicates for the binding of 1μg/ml for each mAb.

Figure 2

Cross-species binding of αPoIFNγ mAbs. A panel of biotin-labeled αPoIFNγ mAbs were tested for their ability to bind purified orthologous rIFNγ proteins from several species as described in M&M. Shown are mean ODs of duplicates for the binding of 1μg/ml for each mAb.

Figure 3

Sandwich ELISA Assay and Quantitation of IL-17A. (A) Comparison of IL-17A standard curves generated with 3 sets of αPoIL-17A mAb pairs as capture vs detection respectively: αPoIL-17A-1.1/2.6; 1.2/1.1; and 2.6/1.1; (B) Detection of rPoIL-17A diluted in PBS-BSA or in pig serum using the anti-PoIL-17A-1.1/2.6 mAb Sandwich assay. The assays were repeated >5 times; (C) Detection of native porcine IL-17A in PBMC supernatants using the anti-PoIL-17A-1.1/2.6 mAb assay. Supernatants were harvested from cells cultured for 24 or 48 hours (24 h/48 h) in medium (BM), PHA or PMA/Iono stimulated cells as described in M&M. This assay was conducted >3 times evidencing repeatability.

Figure 4

Intracellular staining of pig cells with AF647 labeled αPoIL-17A mAbs. Frozen PBMC were cultured overnight before stimulation with BD Leukocytes activation cocktail [containing phorbol myristate acetate (PMA)/ionomycin), and a protein transport inhibitor (Brefeldin A)]. Cells were stained with BD fixable viability stain and Fc receptors were blocked with rabbit serum before surface staining with αCD3 mAb. Cells were then fixed and permeabilized before intracellular staining with each AF647 labeled αPoIL17A mAb. Data were collected using flow cytometry, gating on live lymphocytes and on live CD3+ T cells, and analyzed using FlowJo Software. Shown are staining data for controls (A, B) and 5 new anti-PoIL-17A mAbs (C–G).