Mouse TNF-α ELISA Kit

RACTIVITY	Mouse
SENSITIVITY	<4.5 pg/mL
ASSAY RANGE	15.6-1000 pg/mL
REAGENTS PROVIDED	MOUSE TNF-α MICROTITER PLATE BIOTIN CONJUGATE AVIDIN CONJUGATE MOUSE TNF-α STANDARD  CALIBRATOR DILUENT I CALIBRATOR DILUENT II WASH BUFFER (20X/96 wells, 30X/192 wells)  SUBSTRATE A  SUBSTRATE B  STOP SOLUTION

INTENDED USE 

This Mouse TNF-alpha (α) ELISA Kit is to be used for the in vitro quantitative determination of mouse tumour necrosis factor alpha (mouse TNF-α) concentrations in serum and cell culture supernatant. This kit is intended FOR LABORATORY RESEARCH USE ONLY. 

INTRODUCTION 

Tumor Necrosis Factor alpha (TNF-α) is a multifunctional proinflammatory cytokine, mainly secreted by activated macrophages.  The cytokine was named for its remarkable ability to cause hemorrhagic necrosis of tumors in mice.  It is implicated with a variety of biological procedures including systemic inflammation, cell proliferation, apoptosis, lipid metabolism, and coagulation.  It is well documented that TNF-a functions through its receptors, TNFR1 (p55) and TNFR2 (p75). 

TNF-α plays an important role in the immune response to bacterial, and certain fungal, viral, parasitic invasions, in tissue remodeling, autoimmune-diseases and the necrosis of specific tumors.  The pleiotropic effect of TNF-α regulation is attributed to its ability to trigger multiple signaling pathways simultaneously.  TNF-α hyper-expression in response to the components of some bacteria such as LPS can cause life threatening septic shock.  Recombinant TNF-α, in combination with chemotherapy, has been applied for synergistic treatment of soft sarcomas, melanomas and other irresectable tumors. Anti-TNF-α therapy has been used for treatment of rheumatoid arthritis.

This mouse TNF-α ELISA is a 3.5 hour solid phase immunoassay readily applicable to measure mouse TNF-α levels in serum, plasma, cell culture supernatant in the range of 0 to 1000pg/mL.  

PRINCIPLE OF THE ASSAY

This mouse TNF-α enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay.  The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific to mouse TNF-α.  Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated monoclonal antibody preparation specific for mouse TNF-a and incubated.  Mouse TNF-α 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 mouse TNF-α and other components of the sample.  In order to quantify the amount of mouse TNF-α 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 has 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 mouse 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 mouse TNF-α in the samples, this kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing.)  According to the 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 mouse TNF-α concentration (pg/mL).  The concentration of mouse TNF-α in the samples is then determined by comparing the O.D. of the samples to the standard curve.

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