TNF-a ELISA KIT
INTENDED USE
This Human TNF-a ELISA Kit is to be used for the in vitro quantitative determination of human tumour necrosis factor alpha (TNF-α) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This TNF-a ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and not for use in diagnostic or therapeutic procedures.
INTRODUCTION
Tumour Necrosis Factor alpha (TNF-a), also known as cachectin was initially named for its remarkable ability to cause hemorrhagic necrosis of tumours in mice.The TNF-a gene located on chromosome 6 encodes a 233 amino acid (a.a.) prohormone bound to the plasma membrane, with the mature form (157 a.a., 17 kDa) exposed on the extracellular surface. Soluble, mature TNF-a is released upon cleavage of the C-terminal.The primary source in vivo of TNF-a is thought to be the monocyte/macrophage but various cell types are known to express this cytokine such as lymphocytes, basophils, eosinophils, mast cells, NK cells, T cells, B cells, kerantinocytes, Kupffer cells, astrocytes, and some types of tumours. TNF-α is produced upon stimulation with cytokines such as IL-1, 1L-2, GM-CSF, TNF-a itself and with bacterial lipopolysaccharide (LPS) which is a potent inducer.TNF-a once produced and secreted will bind to TNF-α receptors (TNF-a R1, 55 kDa and TNF-a R2, 75 kDa), located on the plasma membrane of most cells throughout the body except the red blood cell.The two TNF-a receptors deliberate the biological effects of TNF-a. It has been reported that TNF-a R1 is responsible for mediating LPS toxicityand cell cytotoxicityand TNF-a R2 is involved in cellular proliferation.Various pathological conditions are associated with the production of high levels of TNF-a. These include septic shock syndrome, cachexia (e.g. HIV, tuberculosis, cancer), autoimmune diseases, hepatitis, leukemia, myocardial ischaemia, organ transplantation rejection, multiple sclerosis, rheumatoid arthritis, and meningococcal septicemia.Many people die annually from a complication of infectious disease called septic shock syndrome which is triggered by TNF-a. In many cases elevated TNF-a serum levels predict a higher mortality.TNF-a is a pleiotropic cytokine that can induce disease through TNF-a toxicity (tissue injury, catabolic illness, and mediating shock) and improve host defense mechanisms (stimulating inflammation and increasing immune cell function).In the future, therapies may be developed by blocking TNF-a harmful effects and enhancing TNF-a beneficial effects.
INTENDED USE
TNF-a ELISA Kit
TNF-a ELISA Kit is to be used for the in vitro quantitative determination of human tumour necrosis factor alpha (TNF-α) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This TNF-a ELISA Kit is intended FOR LABORATORY RESEARCH USE ONLY and not for use in diagnostic or therapeutic procedures. This TNF-a ELISA Kit is a 4.5 hour solid phase immunoassay readily applicable to measure TNF-α levels in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 2000 pg/mL. It showed no cross reactivity with other cytokines tested. This TNF-a ELISA Kit is expected to be effectively used for further investigations into the relationship between TNF-a and the various conditions mentioned. This TNF-a ELISA Kit applies a technique called a quantitative sandwich immunoassay.
The microtiter plate provided in this TNF-a ELISA Kit has been pre-coated with a monoclonal antibody specific to TNF-a. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TNF-a and incubated. TNF-a if present, will bind and become immobilized by the antibody pre-coated on the wells and then be “sandwiched” by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound TNF-a and other components of the sample. In order to quantify the amount of TNF-a present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each have a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain TNF-α, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
In order to measure the concentration of TNF-a in the samples, this TNF-a ELISA Kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.) According to the TNF-a ELISA Kit testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus TNF-a concentration (pg/mL). The concentration of TNF-a in the samples is then determined by comparing the O.D. of the samples to the standard curve.
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