colorimetric sandwich ELISA kit datasheet For the quantitative detection of rat TNF-alpha concentrations in cell culture supernatants. general information Catalogue Number Product Name Species cross-reactivity Range (calibration Range) Tested applications KE20001 Rat TNF-alpha ELISA Kit Rat TNF-alpha 15.6-1000 pg/ml Quantification ELISA database links Entrez Gene SwissProt 24835 (rat) P16599 (rat) kit components & storage Microplate - antibody coated 96-well Microplate (8 well 12 strips) 1 plate Store at 2-8 C for six months Standard -1000 pg/bottle; lyophilized* 2 bottles Store at 2-8 C for six months Detection antibody, biotinylated (100X) - 120 µl/vial 1 vial Store at 2-8 C for six months Streptavidin-HRP (100X) - 120 µl/vial 1 vial Store at 2-8 C for six months Sample Diluent PT 1-ef - 30 ml/bottle. For cell culture supernatants 1 bottle Store at 2-8 C for six months Detection Diluent - 30 ml/bottle 1 bottle Store at 2-8 C for six months Wash Buffer Concentrate (20X) - 30 ml/bottle 1 bottle Store at 2-8 C for six months Tetramethylbenzidine Substrate (TMB) - 12 ml/bottle 1 bottle Store at 2-8 C for six months Stop Solution - 12 ml/bottle 1 bottle Store at 2-8 C for six months Plate Cover Seals 3 pieces NB: Do not use the kit after the expiration date. Sample Diluent PT 1-ef is for standard and samples. Detection Diluent is for Detection antibody and Streptavidin-HRP. *Add 1 ml Sample Diluent PT1-ef in standard. This reconstitution gives a stock solution of 1000 pg/ml. UK & Europe: Europe@
Add # µl of Standard diluted in the previous step # µl of Sample Diluent PT 1-ef 500 µl 500 µl 500 µl 500 µl 500 µl 500 µl 1000 µl 500 µl 500 µl 500 µl 500 µl 500 µl 500 µl "sd7" "sd6" "sd5" "sd4" "sd3" "sd2" "sd1" product description KE20001 is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (Sandwich ELISA). The TNF-alpha ELISA kit is to be used to detect and quantify protein levels of endogenous TNF-alpha. The assay recognizes rat TNF-alpha. A polyclonal antibody specific for TNF-alpha has been pre-coated onto the microwells. The TNF-alpha protein in samples is captured by the coated antibody after incubation. Following extensive washing, a polyclonal antibody of biotinylated specific for TNF-alpha is added to detect the captured TNF-alpha protein. For signal development, Streptavidin-HRP is added, followed by Tetramethyl-benzidine (TMB) reagent. Solution containing sulfuric acid is used to stop color development and the color intensity which is proportional to the quantity of bound protein is measurable at 450nm with the correction wavelength set at 630 nm. background TNF, as also known as TNF-alpha, or cachectin, is a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. It is expressed as a 26 kda membrane bound protein and is then cleaved by TNF-alpha converting enzyme (TACE) to release the soluble 17 kda monomer, which forms homotrimers in circulation. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. It can bind to, and thus functions through its receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. rat and human TNF-alpha share 79% amino acid sequence identity. Unlike human TNF-alpha, the rat form is glycosylated. In rat deficiency of this gene is associated with defects in response to bacterial infection, with defects in forming organized follicular dendritic cell networks and germinal centers, and with a lack of primary B cell follicles. 2 UK & Europe: Europe@
Optical Density sample preparation The cell culture supernatants may require proper dilution to fall within the range of the assay. A range of dilutions like 1:2, 1:4 is suggested according to the individual samples. safety notes This product is sold for lab research and development use ONLY and not for use in humans or animals. Avoid any skin and eye contact with Stop Solution and TMB. In case of contact, wash thoroughly with water. assay procedure summary Step Reagent Volume Incubation Wash Notes 1 Standard and Samples 100 µl 120 min 4 times Cover Wells 2 Diluent Antibody Solution 100 µl 60 min 4 times Cover Wells 3 Diluent HRP Solution 100 µl 40 min 4 times Cover Wells 4 TMB Substrate 100 µl 15-20 min Do not wash Incubate in the dark at 37 C 5 Stop Solution 100 µl 0 min Do not wash - 6 Read plate at 450 nm and 630 nm immediately after adding Stop solution. DO NOT exceed 5 minutes. typical data These standard curves are provided for demonstration only. A standard curve should be generated for each set of samples assayed. 10 1 0.1 0.01 Four Parameter Logistic (4-PL) Curve Fit 10 100 1000 Rat TNF-α Concentration (pg/ml) R 2 =0.999 (pg/ml) O.D Average Corrected 0 0.083 0.089 0.086-15.6 0.161 0.179 0.170 0.084 31.25 0.236 0.247 0.242 0.156 62.5 0.412 0.405 0.409 0.323 125 0.633 0.651 0.642 0.556 250 1.033 1.039 1.036 0.950 500 1.565 1.679 1.622 1.536 1000 2.255 2.356 2.306 2.220 3 UK & Europe: Europe@
precision Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision. Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 24 24 24 Mean (pg/ml) 288.8 598.9 1088.1 238.2 374.0 716.9 SD 17.9 40.2 79.8 16.9 34.2 37.2 CV% 6.2 6.7 7.3 7.1 9.2 5.2 recovery The recovery of TNF-alpha spiked to three different levels in four samples throughout the range of the assay in cell culture supernatants was evaluated. Sample Type Average% of Expected Range (%) 1:2 93 92-94 Cell culture supernatants 1:4 75 71-77 sensitivity The minimum detectable dose of rat TNF-alpha is 7.7 pg/ml. This was determined by adding two standard deviations to the concentration corresponding to the mean O.D. of 20 zero standard replicates. linearity To assess the linearity of the assay, three samples were spiked with high concentrations of TNF-alpha in cell culture supernatants and diluted with Sample Diluent PT 1-ef to produce samples with values within the dynamic range of the assay. Cell culture supernatants Average% of Expected 105 1:2 Range (%) 102-107 Average% of Expected 99 1:4 Range (%) 94-103 Average% of Expected 96 1:8 Range (%) 95-96 Average% of Expected 90 1:16 Range (%) 89-92 4 UK & Europe: Europe@
references 1. Agbanoma G. et al. (2012) J Immunol. 188: 1307-17. 2. Kriegler M. et al. (1988) Cell. 53: 45-53. 3. Theiss AL. et al. (2005) J Biol Chem. 280: 36099-109. 4. Swardfager W. et al. (2010) Biol Psychiatry. 68:930-41. 5. Locksley RM.et al. (2001) Cell. 104(4):487-501. 6. provided by RefSeq, Jun 2013 5 UK & Europe: Europe@