Dataset Information

Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

ABSTRACT: Tumor necrosis factor-? (TNF?), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNF? can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNF? by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNF? antibody, which may be used as a therapeutic option to inhibit TNF?-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNF?. Four mAbs rescued L929 fibroblast cells from TNF?-triggered cell death and one of these, namely C8, was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNF?-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (glutamine, arginine, glutamic acid) form the core epitope. The observation was supported by bioinformatics analyses of an antigen/antibody complex model. In addition, the binding affinity of mAb C8 to TNF? was found to be comparable with that of infliximab, which is a commercially available anti-TNF? mAb.

SUBMITTER: Jameei A

PROVIDER: S-EPMC6709731 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

Jameei Aida A Nagarajan Deepesh D Sarikhani Mohsen M Chandra Nagasuma N Karande Anjali A AA

Monoclonal antibodies in immunodiagnosis and immunotherapy 20190715 4

Tumor necrosis factor-α (TNFα), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody, which may be used as a therapeutic opti ...[more]

PMID: 31305212

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Project description:The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

Dataset Information

Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

Publications

Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

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