HumanTNF ELISA Kit

RUO, TNF-alpha ELISA isan enzyme-linked immunosorbent assay for quantitative detection of human TNF-alpha in cellculture supernatants, human plasma (EDTA, heparin and citrate), serum, cerebrospinal fluid, urine, synovial fluid or other body fluids. The assaywill recognize both natural and recombinant Hu TNF-alpha.

Tumor Necrosis Factor

NM_000594

191160

cytokine activity, identical protein binding, protease binding, protein binding, transcriptionregulatory region DNA binding, tumor necrosis factor receptor binding

1.Reagent And Sample Preparation 1)Antibody coated plate: Before opening the foil pouch, determine the number ofstrips required to test the desired number of samples, plus 16 wells neededfor running standards and blanks in duplicate. Remove non-used strips fromthe plate-frame and return them to the foil pouch containing the desiccantfor up to 3 months at 2-8oC. 2)Dilution of test standard: Dissolve the lyophilised TNF- standard with SampleDiluent volume shown on the label. TNF- standard is unstable afterdissolving. Use immediately or keep on ice if not used within 1 hr afterdissolving. To obtaina standard curve dilute it as follows: a. Add100 ul of TNF-standard from kit standard tube containing 4000 pg/ml of TNF- and 700 ul to thefirst tube to obtain 500 pg/ml (Standard tube 1.) b. Add150 ul ofSample Diluent to all other 6 dilution tubes. Take 150 ul fromthe first tube (500 pg/ml) and start 2-fold serial dilutions in dilutiontubes as described in the figure by mixing several times with the pipet ineach tube (Total of 7 dilution tubes). c. 150 ul ofsample Diluent in tube 8 serves as zero standard (0 pg/ml). 3) Samplepreparation and dilution: Dilution of samples is not required for initialscreening. Samples that exceed the measuring range should be diluted insample diluent serially 1:2, 1:4, or further if necessary, and measuredagain. The dilution factor must be taken in account when calculating theresults. Diluteand store all samples in tubes or plates made of material with low bindingsurface, such as polypropylene. 4) Samplecollection and storage: Serum, EDTA, heparin or citrate anti-coagulatedplasmas, cerebrospinal fluid, urine, synovial fluid, other body fluids andcell culture supernatants are suitable for use in the assay (caution:separate plasma/serum and blood cells within 4 hours after collection, non-separated samples must be kept at 2-8oC). Do not use grossly haemolysedor lipemic specimens. If samples are to be run within 24 hours, they may bestored at 2-8oC, otherwise samples should be stored frozen (at least between-18 to -32oC, but preferably < -70oC). Up to 3 freeze-thaw cycles have noeffect on the TNF-alpha levels of samples. Nonetheless, excessivefreeze-thaw cycles should be avoided. Prior to the assay, frozen samplesshould be thawed as quickly as possible in tap water (18-25oC), do not use37C or 56C water bath for this purpose. 5)Preparation of reagents: a. WashBuffer: If the 20x concentrated Wash Buffer contains visible crystals, warmit at 37C and mix gently until dissolved. Dilute 1:20 with de-ionized ordistilled water (e.g. 25 ml of Wash Buffer Concentrate and 475 ml distilledwater to yield 500 ml of 1x Wash Buffer). Check the pH of the diluted washbuffer and adjust to 7.4 if necessary. b. Vortexmix Biotinylated antibody solution gently before use. c. Vortexmix peroxidase (HRP) labeled avidin gently before use. 2. AssaySteps 1) Bring allreagents and samples to room temperature (18 - 25oC) before use. It isrecommended that all standards and samples be run at least in duplicate.Leave some wells as a reagent blank (2 to 4 wells). 2) Pipette 50 ul ofsample and 50 ul of eachdiluted standard starting from 500 pg/ml into appropriate wells. Pipette 50 ul ofsample diluent to the wells which will be used as a blank. Incubate 1 hr atroom temperature without shaking. 3) Wash 5x with 1xWash Solution (300 ul each) 4) Promptly add 50 ul ofgreen colored Biotinylated TNF-alpha detection antibody to all wells. Tap theplate gently by hand to homogenize your mixture. Avoid touching to thereaction wells with the pipette tip. 5) Wash 5 times 5xas described in Step 3. 6) Wash 5 times asdescribed in Step 3. 7) Using amultichannel pipette, promptly add 50 ul of TMB ready touse substrate reagent to each well. Incubate for 20 minutes at roomtemperature in the dark. 8) Add 25 ul of StopSolution to each well. Read at 450 nm within 15 minutes. 9) Calculate themean of reagent blank absorbance values and subtract it from all test wellvalues (standard and test samples). Mean reagent blank absorbance value at450 nm should be less than 0.200. 10) Calculate yourresults against standard curve, as outlined below.