Rat TNF alpha ELISpot Assay Kit


This Rat TNF alpha ELISpot Assay Kit is designed to determine the frequency of TNF alpha producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The Eagle Biosciences Rat TNF alpha ELISpot Assay Kit is for research use only and not to be used for diagnostic procedures.

Rat TNF alpha ELISpot Assay Kit

The Rat TNF alpha ELISpot Assay Kit is for research use only.

Incubation Time: 18hrs – 23hrs
Sample Size: 100 µl

Assay Principle

The ELISpot is a highly specific immunoassay for the analysis of cytokine and other soluble molecule production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation. This technique is designed to determine the frequency of cytokine producing cells under a given stimulation and the comparison of such frequency against a specific treatment or pathological state. The ELISpot assay constitutes an ideal tool in the investigation of Th1 / Th2 responses, vaccine development, viral infection monitoring and treatment, cancerology, infectious disease, autoimmune diseases and transplantation. Utilizing sandwich immuno-enzyme technology, ELISpot assays can detect both secreted cytokines and single cells that simultaneously produce multiple cytokines. Cell secreted cytokines or soluble molecules are captured by coated antibodies avoiding diffusion in supernatant, protease degradation or binding on soluble membrane receptors. After cell removal, the captured cytokines are revealed by tracer antibodies and appropriate conjugates. A capture antibody highly specific for the analyte of interest is coated to the wells of a PVDF bottomed 96 well microtiter plate either during kit manufacture or in the laboratory. The plate is then blocked to minimize any non-antibody dependent unspecific binding and washed. Cell suspension and stimulant are added and the plate incubated allowing the specific antibodies to bind any analytes produced. Cells are then removed by washing prior to the addition of Biotinylated detection antibodies which bind to the previously captured analyte. Enzyme conjugated streptavidin is then added binding to the detection antibodies. Following incubation and washing substrate is then applied to the wells resulting in colored spots which can be quantified using appropriate analysis software or manually using a microscope.

Related Products

TNF-Alpha ELISA Assay Kit
TNF alpha ELISpot Assay Kit

Additional Information

Assay Background

Tumor Necrosis Factor (TNFα), also known as cachectin, is a polypeptide cytokine produced by monocytes and macrophages. It functions as a multipotent modulator of immune response and further acts as a potent pyrogen. TNFα circulates throughout the body responding to stimuli (infectious agents or tissue injury), activating neutrophils, altering the properties of vascular endothelial cells, regulating metabolic activities of other tissues, as well as exhibiting tumoricidal activity by inducing localized blood clotting. TNFα also inhibits lipoprotein lipase activity resulting in cachexia, a physical wasting condition. Activation of B-cells by the Epstein Barr virus can be inhibited by TNFα. Due to its varied actions throughout the immune system, TNFα may play a role in the pathogenesis of many disease states. TNFα production is mediated by the action of lymphokines and endotoxins on the macrophage. Purified monocytes produce TNFα within four hours of stimulation by recombinant IL-2 and there is some in vitro evidence to suggest that TNFα is expressed at high levels and with prolonged kinetics in T cells stimulated by both CD2 and CD28. Secretion of TNFα is enhanced by gamma interferon. TNFα then induces or enhances the specific production of Class I MHC antigen, GM-CSF, and IL-1. Recent evidence has suggested an intracellular role for this peptide. TNFα may play a significant role in the pathogenesis of inflammatory disease of the joints and other tissues. Chin et al. found that TNFα, along with gamma interferon and IL-1 increased cell surface expression of ICAM-1 on synovial fibroblasts. Alvaro-Garcia et al. reported that TNFα stimulates synovial proliferation. Waage et al. found that increased levels of TNFα in patients with septicemia and meningococcal disease correlated with fatal outcome. Scuderi et al. suggest that increased levels of this cytokine may play a role in the host defense mechanism against parasitic infections. Girardin et al. reported that increased serum TNFα levels correlated with the number of risk factors involved in children with gram-negative sepsis and purpura fulminians. Elevated levels of TNFα were also found in individuals suffering from myocarditis. Recently, a growing body of information has pointed to a role for TNFα in the pathogenesis of AIDS. Alveolar macrophages (AM) from HIV positive individuals with opportunistic lung infections have been shown to spontaneously produce higher levels of TNFα in vitro than those HIV positive individuals without infection and HIV negative controls. Krishnan et al. report that higher TNFα production by AM was associated with lower counts of pneumocystis carinii in broncheoalveolar lavage fluid, indicating that TNFα may play a role in the control of this infection in AIDS. Israel-Biet et al. also reported in in-vitro studies, that AM that express HIV (p24+) released significantly higher levels of TNFα than p24- alveolar macrophages and controls. Reddy et al. found persistently elevated levels of circulating TNFα in HIV seropositive individuals and suggest a possible involvement of this cytokine in the development of AIDS. Measurement of TNFα levels has also been shown to be useful in transplant research, where Maury et al. and McLaughlin et al. Both reported TNFα to be markedly elevated in renal allograft rejection episodes. Recent evidence has been presented on increased TNFα levels in Bone Marrow Transplant (BMT). BMT patients with major transplant related complications such as interstitial pneumonitis and severe acute graft-versus – host disease had TNFα levels significantly increase over controls.

ELISpot Assay Kit Procedure


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