Human VEGFA ELISA Kit

RUO, Vascular endothelialgrowth factor (VEGF) ELISA is an enzyme-linked immunosorbent assay for thequantitative detection of human VEGF levels in cell culture supernatants, human serum, plasma (EDTA, heparin and citrate), CSF and urine.

vascular endothelial growth factor A

NM_003376

192240

cell surfacebinding, chemoattractant activity, cytokine activity, cytokine activity, extracellular matrix binding, fibronectin binding, growth factor activity, growth factor activity, heparin binding, heparin binding, platelet-derivedgrowth factor receptor binding, protein heterodimerization activity, proteinhomodimerization activity, vascular endothelial growth factor receptor 1binding, vascular endothelial growth factor receptor 2 binding, vascularendothelial growth 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: a. Take100 ul ofVEGF165 from kit standard tube containing 4200 pg/ml of VEGF and pipette intoStandard tube 1. Add 500 ul of sample diluent to obtain VEGFconcentration of 700 pg/ml in the first dilution tube (total volume 600 ul). b. Add200 ul ofSample Diluent to the other 4 dilution tubes. Take 100 ul fromthe first tube (700 pg/ml) and start 3-fold serial dilutions in dilutiontubes as described in the figure by mixing several times with the pipette ineach tube (Total of 5 dilution tubes). c. 200 ul ofsample Diluent serves as zero standard (0 ng/ml) in tube 6. 3) Samplepreparation and dilution: Dilution of samples is not required for initialscreening. Samples that exceed the measuring range should be diluted seriallyin sample diluent and measured again. Samples with absorbance values>1.900 can be serially diluted 1:2, 1:4, 1:8, or further if necessary. Thedilution factor must be taken in account when calculating the results. 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, cell culture supernatants and urine aresuitable sample types for use in the assay (caution: separate plasma/serumand blood cells within 4 hours after collection, non-separated samples mustbe kept in temperatures from +2-8oC). Do not use grossly haemolyzed orlipemic 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 VEGF levels of samples. Nonetheless, excessive freeze-thawcycles should be avoided. Prior to the assay, frozen samples should be thawedas quickly as possible in tap water (18-25oC). Do not use 37C or 56C waterbath 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 green colored Biotinylated antibody solution gently before use. c. Vortexmix blue colored peroxidase (HRP) labeled avidin gently before use. 2. AssaySteps 1) Bringall reagents and samples to room temperature (18 - 25oC) before use. It isrecommended that all standards and samples are 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 1200 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 5times with 1x Wash Solution (300 ul each) 4)Promptly add 50 ul ofgreen colored Biotinylated VEGF detection antibody to all wells. Tap theplate gently by hand to homogenize your mixture. Avoid touching the reactionwells with the pipette tip. 5) Wash 5times as described in Step 3. 6) Wash 5times as described in Step 3. 7) Usinga multichannel pipet, promptly add 50 ul of TMB ready touse Substrate Solution to each well. Incubate for 20 minutes at roomtemperature in the dark. 8) Add 25ul of StopSolution to each well. Read at 450 nm within 15 minutes. 9)Calculate the mean of reagent blank absorbance values and subtract it from alltest well values (standard and test samples). Mean reagent blank absorbancevalue at 450 nm should be less than 0.200. 10)Calculate your results against standard curve, as outlined below.