Development of monoclonal antibodies for quantification of bovine tumor necrosis factor-α

Abstract

Highlights• Tumor necrosis factor-α (TNF-α) is a key mediator of early inflammation in dairy cows.• We generated 2 monoclonal antibodies specific to bovine TNF-α.• These monoclonal antibodies can be used to quantify soluble TNF-α in a bead-based assay.• In flow cytometric application, they can differentiate cytoplasmatic TNF-α in leukocytes.

Development of a bead-based multiplex assay to quantify bovine interleukin-10, tumor necrosis factor-α, and interferon-γ concentrations in plasma and cell culture supernatant

“…We chose the following mAbs for coupling to different fluorescent beads (MicroPlex Microspheres, Luminex Corp.): anti-bovine TNF-α 197-1 (IgG 1 ; Sipka et al, 2021) to bead 32; anti-bovine IL-10 CC318 (IgG 2b , MCA2110, BioRad; Kwong et al, 2002) to bead 33; and anti-bovine IFN-γ CC330 (IgG 1 , MCA2112, BioRad; Baszler et al, 2008) to bead 35. All coupling and detection mAbs were developed in mice.…”

“…The bead-coupled mAbs were then incubated with blocking buffer (PBN, PBS plus 1% BSA and 0.05% sodium azide) for 30 min at room temperature, washed 3 times with PBS plus 0.1% BSA, 0.05% sodium azide, and 0.02% Tween 20 (PBS-T), counted in a Neubauer hemocytometer, and stored at 4°C in the dark. The following biotinylated mAbs were used as detection antibodies in the assay: anti-bovine TNF-α 65-2 (IgG 1 ; Sipka et al, 2021), anti-equine IL-10 492-2 (IgG 1 ; Wagner et al, 2008), and anti-bovine IFN-γ CC302 (IgG 1 , MCA1783B, BioRad; Johnson et al, 2008). Specificity of the mAbs used in the assay was previously described by our group or others.…”

“…The antibody pair detecting TNF-α was previously shown to selectively detect cytoplasmatic protein in CD14 + mononuclear cells after in vitro stimulation with LPS. Detection in lymphoid cell populations was only observed after PMA and ionomycin stimulation (Sipka et al, 2021). Each of the antibodies was described to bind to the native protein under experimental conditions involving bovine leukocyte populations expected to express the respective cytokine.…”

“…Cells were transfected with mammalian expression vectors (pcDNA3.1, Invitrogen) containing the coding sequence of each bovine cytokine linked to the constant heavy chain region gene of equine IgG 1 (IGHG1) as previously described (Wagner et al, 2005). The coding sequences (cDNA) for bovine TNF-α (GenBank: NM173966.3, bases 194-898), bovine IL-10 (GenBank: EU276074.1, bases 1-537), and bovine IFN-γ (GenBank: EU276066.1, bases 24-524) were amplified from bovine peripheral blood mononuclear cells (PBMC) and cloned into the expression vector as previously described (Sipka et al, 2021).…”