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Rapid and sensitive assay of tumor necrosis factor-x gene transcription.

Zhang X; Ye J; Wang L; Manosroi J; Shi X; Rojanasakul Y
Pharm Res 2001 Mar; 18(3):408-411
Tumor necrosis factor-alpha (TNF-a) is a cytokine that appears rapidly in response to infection and trauma. It plays a beneficial role as an immunostimulant and important mediator of host defense against infectious agents and malignant tumors. However, when abnormally regulated, i.e., because of an autoimmune response or tissue injury, TNF-a can cause severe systemic toxicity and even death. High levels of TNF-a have been associated with various pathological states, such as cachexia and sepsis, and transgenic animals overexpressing TNF-a develop pathological inflammatory conditions, including polyarthritis and central nervous system demyelination. Moreover, neutralization of TNF-a activity leads to improvement in models of inflammatory diseases and in patients with arthritis and sepsis. Because of the physiologic and pathologic importance of TNF-a, several TNF-a-modulating drugs have been developed for therapeutic purposes. Major therapeutic strategies developed thus far have been focused on TNF-a antibodies and antagonists. Antibodies have several advantages, such as target specificity over other therapeutic agents; however, their use has been largely limited resulting from their potential immunotoxicity. Although great efforts have been made to humanize the antibodies to avoid their immunogenicity, several alternative strategies, including those using small non-immunogenic molecules, increasingly have been investigated as antibody replacements. A major problem concerning the development of TNFa- modulating drugs has been the lack of rapid and effective assays for TNF-a. Traditional bioassays and enzyme-linked immunosorbent assay (ELISA), although sensitive, are timeconsuming and cost-prohibitive, especially when a large number of drug candidates need to be tested. In addition, these assays do not permit evaluation of drug's action at the gene transcription level in which most developing TNF-a drugs act. Therefore, there is a need for a transcription-specific, more morerapid assay for TNF-a. In this study, we report a new, highly sensitive and specific assay for TNF-a gene transcription. This assay is based on the use of TNF reporter cell line, which was created by stable transfection of a macrophage cell line (RAW264.7) with a TNF-luciferase plasmid vector. The macrophage cell line was chosen because macrophages are the primary source of TNF-a production in the body. This reporter cell line provides several potential advantages over existing methods, which include: 1) it can be propagated or maintained indefinitely and is therefore always available when needed; 2) it allows easy automation and high throughput applications, i.e., through the use of 96-well plates; and 3) it permits simultaneous detection of TNF-a gene transcription and protein expression. Our results showed that this reporter cell line was sensitive to TNF-a activation and provided test results that correlate well with the ELISA results.
Infectious-diseases; Tumors; Toxic-effects; Models; Diseases; Therapeutic-agents; Antibody-response; Bioassays; Immunology; Immune-reaction; Author Keywords: TNF-a; gene transcription; assay; transfection
Department of Pharmaceutical Sciences, West Virginia University School of Pharmacy, P.O. Box 9530, Morgantown, West Virginia, 26506
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Page last reviewed: July 16, 2021
Content source: National Institute for Occupational Safety and Health Education and Information Division