Bovine TNF alpha (TNFSF2) Do-It-Yourself ELISA, ≤10 Plates

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TNFα (TNFSF2) is a member of the TNF Superfamily. It is produced chiefly by activated macrophages, but it is produced also by a broad variety of cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipose tissue, fibroblasts, and neuronal tissue. The primary role of TNFα is in the regulation of immune cells. TNFα, being an endogenous pyrogen, is able to induce fever, to induce apoptotic cell death, to induce sepsis (through IL-1 & IL-6 production), to induce cachexia, induce inflammation, and to inhibit tumorigenesis and viral replication.

The Bovine TNFα (TNFSF2) Do-It-Yourself ELISA contains capture antibody, protein standard, and detection antibody for development of a Bovine TNFα ELISA. The antibodies have been determined to function in an ELISA with the standard provided. Optimal buffers, concentrations, incubation times, incubation temperatures, and methods for the ELISA have not been determined. A working knowledge of ELISA is strongly recommended. The quantities of components provided are not matched. Components may also be purchased separately. 

The Bovine TNFα (TNFSF2) Do-It-Yourself ELISA can also be used to measure Bison, Water Buffalo, Wild Yak, and Zebu TNFα.

For additional tips and techniques to ensure a successful ELISA, check out our ELISA Technical Guide.

Component Usage Quantity Catalog #
Anti-Bovine TNFα Polyclonal Antibody Capture Antibody 100 µg PB0275B-100
Biotinylated Anti-Bovine TNFα Polyclonal Antibody Detection Antibody 50 µg PBB0278B-050
Bovine TNFα Recombinant Protein Standard 5 µg RP0055B-005


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1 Pack
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Measurement of Bovine, Bison, Water Buffalo, Wild Yak, and Zebu TNF alpha in an ELISA.


A TLR7 agonist activates bovine Th1 response and exerts antiviral activity against bovine leukemia virus.

Sajiki Y, Konnai S, Okagawa T, Maekawa N, Nakamura H, Kato Y, Suzuki Y, Murata S, Ohashi K.

Dev Comp Immunol. 2020 Sep 2;114:103847. doi: 10.1016/j.dci.2020.103847.

Applications: Measurement of bovine TNF alpha, IFN alpha, and IFN gamma in culture supernatants by ELISA


Lactation stage impacts the glycolytic function of bovine CD4+ T cells during ex vivo activation.

Eder JM, Gorden PJ, Lippolis JD, Reinhardt TA, Sacco RE.

Sci Rep. 2020 Mar 4;10(1):4045. doi: 10.1038/s41598-020-60691-2.

Applications: Measurement of bovine TNF alpha, IL-2, and IFN gamma in culture supernatants by ELISA


Dairy cattle undergo dynamic physiological changes over the course of a full lactation into the dry period, which impacts their immunocompetence. During activationT cells undergo a characteristic rewiring to increase the uptake of glucose and metabolically reprogram to favor aerobic glycolysis over oxidative phosphorylation. To date it remains to be completely elucidated how the altered energetic demands associated with lactation in dairy cows impacts T cell metabolic reprogramming. Thus, in our ex vivo studies we have examined the influence of stage of lactation (early lactation into the dry period) on cellular metabolism in activated bovine CD4+ T cells. Results showed higher rates of glycolytic function in activated CD4+ T cells from late lactation and dry cows compared to cells from early and mid-lactation cows. Similarly, protein and mRNA expression of cytokines were higher in CD4+ T cells from dry cows than CD4+ T cells from lactating cows. The data suggest CD4+ T cells from lactating cows have an altered metabolic responsiveness that could impact the immunocompetence of these animals, particularly those in early lactation, and increase their susceptibility to infection.


Effect of prepartum energy intake and supplementation with ruminally protected choline on innate and adaptive immunity of multiparous Holstein cows.

Zenobi MG, Gardinal R, Zuniga JE, Mamedova LK, Driver JP, Barton BA, Santos JEP, Staples CR, Nelson CD.

J Dairy Sci. 2020 Mar;103(3):2200-2216. doi: 10.3168/jds.2019-17378. Epub 2020 Jan 15.

Applications: Measurement of bovine TNF alpha in cull culture supernatant by ELISA


Inflammation and oxidative stress transcription profiles due to in vitro supply of methionine with or without choline in unstimulated blood polymorphonuclear leukocytes from lactating Holstein cows.

Lopreiato V, Vailati-Riboni M, Bellingeri A, Khan I, Farina G, Parys C, Loor JJ.

J Dairy Sci. 2019 Nov;102(11):10395-10410. doi: 10.3168/jds.2019-16413. Epub 2019 Aug 22.

Applications: Measurement of bovine TNF alpha in culture supernatants by ELISA


Neutrophils are the most important polymorphonuclear leukocytes (PMNL), representing the front-line defense involved in pathogen clearance upon invasion. As such, they play a pivotal role in immune and inflammatory responses. Isolated PMNL from 5 mid-lactating Holstein dairy cows were used to evaluate the in vitro effect of methionine (Met) and choline (Chol) supplementation on mRNA expression of genes related to the Met cycle and innate immunity. The target genes are associated with the Met cycle, cell signaling, inflammation, antimicrobial and killing mechanisms, and pathogen recognition. Treatments were allocated in a 3 × 3 factorial arrangement, including 3 Lys-to-Met ratios (L:M, 3.6:1, 2.9:1, or 2.4:1) and 3 levels of supplemental Chol (0, 400, or 800 μg/mL). Three replicates per treatment group were incubated for 2 h at 37°C and 5% atmospheric CO2. Both betaine-homocysteine S-methyltransferase and choline dehydrogenase were undetectable, indicating that PMNL (at least in vitro) cannot generate Met from Chol through the betaine pathway. The PMNL incubated without Chol experienced a specific state of inflammatory mediation [greater interleukin-1β (IL1B), myeloperoxidase (MPO), IL10, and IL6] and oxidative stress [greater cysteine sulfinic acid decarboxylase (CSAD), cystathionine gamma-lyase (CTH), glutathione reductase (GSR), and glutathione synthase (GSS)]. However, data from the interaction L:M × Chol indicated that this negative state could be overcome by supplementing additional Met. This was reflected in the upregulation of methionine synthase (MTR) and toll-like receptor 2 (TLR2); that is, pathogen detection ability. At the lowest level of supplemental Chol, Met downregulated GSS, GSR, IL1B, and IL6, suggesting it could reduce cellular inflammation and enhance antioxidant status. At 400 µg/mL Chol, supplemental Met upregulated PMNL recognition capacity [higher TLR4 and L-selectin (SELL)]. Overall, enhancing the supply of methyl donors to isolated unstimulated PMNL from mid-lactating dairy cows leads to a low level of PMNL activation and upregulates a cytoprotective mechanism against oxidative stress. Enhancing the supply of Met coupled with adequate Chol levels enhances the gene expression of PMNL pathogen-recognition mechanism. These data suggest that Chol supply to PMNL exposed to low levels of Met effectively downregulated the entire repertoire of innate inflammatory-responsive genes. Thus, Met availability in PMNL during an inflammatory challenge may be sufficient for mounting an appropriate biologic response.


Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Putz EJ, Eder JM, Reinhardt TA, Sacco RE, Casas E, Lippolis JD.

J Dairy Sci. 2019 Oct;102(10):9268-9284. doi: 10.3168/jds.2019-16448. Epub 2019 Aug 7.

Applications: Measurement of bovine TNF alpha in serum and skim milk by ELISA


Aerosol vaccination with Bacille CalmetteGuerin induces a trained innate immune phenotype in calves.

Guerra-Maupome M, Vang DX, McGill JL.

PLoS One. 2019 Feb 22;14(2):e0212751. doi: 10.1371/journal.pone.0212751. eCollection 2019.

Applications: Measurement of bovine TNF alpha, IL-1 beta, and IL-6 in culture supernatants by ELISA


Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine for use against tuberculosis (TB); however, it is known to reduce childhood mortality from infections other than TB. The unspecific protection induced by BCG vaccination has been associated with the induction of memory-like traits of the innate immune system identified as 'trained' immunity. In humans and mouse models, in vitro and in vivo BCG training leads to enhanced production of monocyte-derived proinflammatory cytokines in response to secondary unrelated bacterial and fungal pathogens. While BCG has been studied extensively for its ability to induce innate training in humans and mouse models, BCG's nonspecific protective effects have not been defined in agricultural species. Here, we show that in vitro BCG training induces a functional change in bovine monocytes, characterized by increased transcription of proinflammatory cytokines upon restimulation with the toll-like receptor agonists. Importantly, in vivo, aerosol BCG vaccination in young calves also induced a 'trained' phenotype in circulating peripheral blood mononuclear cells (PBMCs), that lead to a significantly enhanced TLR-induced proinflammatory cytokine response and changes in cellular metabolism compared to PBMCs from unvaccinated control calves. Similar to the long-term training effects of BCG reported in humans, our results suggest that in young calves, the effects of BCG induced innate training can last for at least 3 months in circulating immune populations. Interestingly, however, aerosol BCG vaccination did not 'train' the innate immune response at the mucosal level, as alveolar macrophages from aerosol BCG vaccinated calves did not mount an enhanced inflammatory response to secondary stimulation, compared to cells isolated from control calves. Together, our results suggest that, like mice and humans, the innate immune system of calves can be 'trained'; and that BCG vaccination could be used as an immunomodulatory strategy to reduce disease burden in juvenile food animals before the adaptive immune system has fully matured.


Choline Regulates the Function of Bovine Immune Cells and Alters the mRNA Abundance of Enzymes and Receptors Involved in Its Metabolism in vitro.

Garcia M, Mamedova LK, Barton B, Bradford BJ.

Front Immunol. 2018 Oct 25;9:2448. doi: 10.3389/fimmu.2018.02448. eCollection 2018.

Applications: Measurement of bovine TNF alpha in cull culture supernatant by ELISA


Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne’s Disease.

Sajiki Y, Konnai S, Okagawa T, Nishimori A, Maekawa N, Goto S, Ikebuchi R, Nagata R, Kawaji S, Kagawa Y, Yamada S, Kato Y, Nakajima C, Suzuki Y, Murata S, Mori Y, Ohashi K.

Infect Immun. 2018 Apr 23;86(5):e00910-17. doi: 10.1128/IAI.00910-17. Print 2018 May.

Applications: Measurement of bovine TNF alpha in culture supernatants by ELISA


Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.


Bovine leukemia virus reduces anti-viral cytokine activities and NK cytotoxicity by inducing TGF-β secretion from regulatory T cells.

Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi K.

Immun Inflamm Dis. 2016 Jan 18;4(1):52-63. doi: 10.1002/iid3.93. eCollection 2016 Mar.

Applications: IL-2 was used to to stimulate several different cell types; TNF alpha was measured in cell culture supernatants by ELISA


CD4(+)CD25(high)Foxp3(+) T cells suppress excess immune responses that lead to autoimmune and/or inflammatory diseases, and maintain host immune homeostasis. However, CD4(+)CD25(high)Foxp3(+) T cells reportedly contribute to disease progression by over suppressing immune responses in some chronic infections. In this study, kinetic and functional analyses of CD4(+)CD25(high)Foxp3(+) T cells were performed in cattle with bovine leukemia virus (BLV) infections, which have reported immunosuppressive characteristics. In initial experiments, production of the Th1 cytokines IFN-γ and TNF-α was reduced in BLV-infected cattle compared with uninfected cattle, and numbers of IFN-γ or TNF-α producing CD4(+) T cells decreased with disease progression. In contrast, IFN-γ production by NK cells was inversely correlated with BLV proviral loads in infected cattle. Additionally, during persistent lymphocytosis disease stages, NK cytotoxicity was depressed as indicated by low expression of the cytolytic protein perforin. Concomitantly, total CD4(+)CD25(high)Foxp3(+) T cell numbers and percentages of TGF-β(+) cells were increased, suggesting that TGF-β plays a role in the functional declines of CD4(+) T cells and NK cells. In further experiments, recombinant bovine TGF-β suppressed IFN-γ and TNF-α production by CD4(+) T cells and NK cytotoxicity in cultured cells. These data suggest that TGF-β from CD4(+)CD25(high)Foxp3(+) T cells is immunosuppressive and contributes to disease progression and the development of opportunistic infections during BLV infection.

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