R&D Systems Quantikine solid phase ELISA is designed to measure the cytokine TNF-alpha in cell culture supernatants, serum and plasma. The assay takes between 3.5-4.5 hours depending on the starting material and uses a monoclonal antibody specific for TNF-alpha pre-coated onto a 96-well, individual strip microplate. Standards (recombinant human TNF-alpha) and samples are added to the wells and any TNF-alpha present is bound by the immobilized antibody. After washing the microplate to remove any unbound substances, an enzyme-linked (HRP) polyclonal antibody specific for TNF-alpha is added to the microplate wells. The microplate is washed once again to remove any unbound antibody-enzyme conjugate. At this point, the substrate solution is added to the wells and color development is allowed to proceed (develops in proportion to the amount of TNF-alpha bound in the initial step). For analysis, the color development is stopped and the intensity is measured.
We have used this assay primarily to measure levels of TNF-alpha produced by monocyte cell lines after mitogen stimulation. All reagents are brought to room temperature before beginning the assay. The first step in the ELISA is to prepare the standards in the supplied calibrator diluent (RD6-35). When using cell culture supernatants, the calibrator diluent needs to be diluted itself (1:5) with deionized or distilled water. The recombinant TNF-alpha standard is reconstituted in 1 ml of deionized or distilled water to make a 10000 pg/ml solution and allowed to incubate at room temperature, with gentle agitation, for 15 min. Once the TNF-alpha is made, dilutions are prepared in RD6-35 to give TNF-alpha standards from 1000-15.6 pg/ml in doubling dilutions. The ELISA plate is removed from its foil pouch and the assay diluent (RD1F) is added to each well (50 ul). At this point, 200 ul of each standard (in duplicate) and the samples are added to the plate and incubated for 2 h at room temperature. After the incubation, the plate is aspirated and washed four times with the supplied wash buffer. Following washing, 200 ul of TNF-alpha conjugate (polyclonal anti-TNF-alpha-HRP) is added to the plate and a further incubation of 1 h at room temperature is performed (2 h incubation if measuring serum or plasma samples). Once again, after the incubation the plate is aspirated and washed 4 times. The next step is to add 200 ul of prepared substrate solution to the plate (equal volumes of color reagents A and B), incubate for 20 min while the color develops (blue) and add the stop solution upon completion. Although 20 min is the recommended incubation time, it is important to check the plate in order to prevent over development of the color. The plate can be read in a microplate reader at 450 nm as the stop solution turns the blue color to yellow.
A standard curve is created from the standards on the plate and is used to calculate the concentration of TNF-alpha in the samples. There are many types of software which will perform this step; we have used Molecular Devices Softmax pro. It is important to keep the level of TNF-alpha within the dynamic range of the assay possibly requiring optimization on the first use of the kit. We have found the intra- and inter assay precision to be very robust.
Boyd Scott
Senior Research Scientist
Discovery Biology
Vitae Pharmaceuticals