ACE2 (human) ELISA Kit
Contents
1. at 37 C Remove plate from 37 C aspirate and wash x 5 with 300 ul of 1X Wash Buffer After last wash tap inverted plate on a stack of paper towels Complete removal of liquid is essential for good performance Add 100 ul of TMB Substrate Solution Allow the color to develop at room temperature in the dark for 30 min Stop the reaction by adding 100 ul of Stop Solution to each well Tap the plate gently to ensure thorough mixing The substrate reaction yields a blue solution that turns yellow when Stop Solution is added Caution Stop Solution is a Corrosive Solution Measure the OD at 450 nm in an ELISA plate reader within 30 min Fax 408 493 1801 tech biovision com Tel 408 493 1800 www biovision com Page 1 of 2 BioVision V Calculations 1 Average the duplicate readings for each standard QC and sample and subtract the the recovery averages 95 range from 81 to 113 average blank value obtained with the 0 ng ml point 2 Generate the standard curve by plotting the average absorbance obtained for each Samples Average Recovery standard concentration on the horizontal X axis vs the corresponding ACE2 a a gt concentration ng ml on the vertical Y axis see 10 TYPICAL DATA 3 Calculate the ACE2 concentrations of samples by interpolation of the regression curve formula as shown above in a form of a quadratic equation 4 If the test samples were diluted multiply the interpolated values by the dilu2. BioVision ACE2 human ELISA Kit Catalog K4918 100 100 assays Store kit at 4 C Description By EST database searching for sequences showing homology to the zinc metalloprotease angiotensin converting enzyme a full length ACE2 cDNA originally called ACEH was isolated which encoded a deduced 805 amino acid protein that shares approximately 40 identity with the N and C terminal domains of ACE ACE2 contains a potential 17 amino acid N terminal signal peptide and a putative 22 amino acid C terminal membrane anchor Northern blot analysis detected high expression of ACE2 in kidney testis and heart and moderate expression in colon small intestine and ovary The soluble truncated form of ACE2 lacks the transmembrane and cytosolic domains in CHO cells and is glycosylated protein that was able to cleave angiotensin and angiotensin Il but not bradykinin ACE converts angiotensin to angiotensin II which has 8 amino acids ACE2 converts angiotensin to angiotensin 1 9 which has 9 amino acids This can then further be converted by ACE to a shorter peptide angiotensin 1 7 which is a blood vessel dilator Using ACE2 null mice Crackower et al showed that ACE2 was critically involved in a cardiac contractility This assay is a sandwich Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of human ACE2 in biological fluids A polyclonal antibody specific for ACE2 has been precoated onto the 96 well microtiter pla
3. N n Washes too stringent Use an automated plate washer if possible Incubation times Incubation times should be followed as OAOT RAR optimal plate reader 6 45697 3724 8149 Use recommended incubation temperature 2 Inter assay precision Six samples of known concentrations of human ACE2 were USE assayed in 11 separate assays to test precision between assays N n rev 02 12 For research use only 3 Recovery When samples urine are spiked with known concentrations of human ACE2 Concentration of a detector too high Use recommended dilution factor High background Inadequate washing Ensure all wells are filling wash buffer and are aspirated completely Wells not completely Poor standard aspirated Completely aspirate wells between steps i Reagents poorly mixed Be sure that reagents are thoroughly mixed Be sure that reagents were prepared correctly Unexpected Omissionmanioadents and added in the correct order results ipetti j 2 Diuiioirertor Check pipetting technique and double check calculations Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com 6 49219 3 144 6 388 FOR RESEARCH USE ONLY Not to be used on humans BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Page 2 of 2
4. X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1X Diluent 300 pl 300 l 300ul 6 3300p 300 300pl 300 MX MXMX MOO 5 ng ml Empty tube 25 12 5 6 25 ng ml ng ml ng ml 3 125 ng ml 1 563 ng ml 0 781 ng ml 0 391 0 ng ml ng ml 2 Reagent Preparation Prepare just the appropriate amounts for the assay 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer 1X Diluent Dilute 5X Wash Buffer 1 4 with dH2O to obtain 1X Diluent 1X Detector 100X HRP Labeled Streptavidin Dilute 100X Detector 1 99 with 1X Diluent to obtain 1X Detector Note The diluted Detector must be used within 1 hr of preparation 3 Assay Protocol Determine the number of 8 well strips needed for assay and insert them into the frame for current use The extra strips should be resealed in the foil pouch and can be stored at 4 C for up to 1 month Add 100 ul of the Standards Samples and QC Sample into the appropriate wells in duplicate Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash x 3 with 300 ul of 1X Wash Buffer Warm Detection Antibody to room temperature Add 100 ul to each well and tap genily on the side of the plate to mix Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash x 3 with 300 ul of 1X Wash Buffer Add 100 ul of the 1X Detector to each well Cover plate with plate sealer and incubate for 1 hr
5. greater than 25 C Assay Procedure Read the ENTIRE Protocol Before Proceeding Test Samples Standards QC Sample We recommend these be run in duplicate a Urine Aseptically collect the urine of the day voided directly into a sterile container Assay immediately or aliquot and store at lt 20 C Avoid repeated freeze thaw cycles b Cell Culture Supernatant has to be diluted in Diluent 1X Samples containing visible precipitates must be clarified before use NOTE As a Starting point 1 2 dilution of urine is recommended If samples fall the outside range of assay a lower or higher dilution may be required c QC Sample Reconstitute Human ACE2 QC sample with 1 ml of dH20 Mix the QC Sample to ensure complete reconstitution Allow to sit for a minimum of 15 min The QC Sample is ready to use do not dilute it refer to the C of A for current QC Sample concentration BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA d rev 02 12 For research use only Standards Reconstitute human ACE2 Standard with 1 ml of dH2O to produce a stock solution 50 ng ml Mix the Stock solution to ensure complete reconstitution Allow to sit for a minimum of 15 min The reconstituted standard should be aliquoted and stored at 20 C Prepare 1X Diluent Dilute 5X Diluent 1 4 with dH2O Prepare Standard Curve using 2 fold serial dilutions with 1X Diluent Toobtain_ A 300 ul of 1X Diluent 300 ul of 1X Diluent 300 ul of 1
6. te Standards and samples are pipetted into the wells for binding to the coated antibody After extensive washing to remove unbound compounds ACE2 is recognized by the addition of a biotinylated polyclonal antibody specific for ACE2 Detection Antibody After removal of excess biotinylated antibody HRP labeled streptavidin Detector is added Following a final washing peroxidase activity is quantified using the substrate 3 3 5 5 tetramethylbenzidine TMB The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of ACE2 in the samples The assay range is 0 391 25 ng ml ACE2 ml The lowest level of ACE2 that can be detected by this assay is 293 pg ml Kit Contents 100 Assays Part Number Component 1 plate coated with human ACE2 Antibody 12 x 8 well strips K4918 100 1 50 ml K4918 100 2 1 bottle Wash Buffer 10X 1 bottle Diluent 5X K4918 100 3 1 bottle Detection Antibody K4918 100 4 K4918 100 5 K4918 100 6 K4918 100 7 K4918 100 8 K4918 100 9 K4918 100 10 1 vial Detector 100X HRP Labeled Streptavidin 1 vial human ACE2 Standard lyophilized 1 vial human ACE2 QC sample lyophilized 1 bottle TMB Substrate Solution 1 bottle Stop Solution 3 plate sealers plastic film 12 x 8 well strips Storage Conditions Reagents must be stored at 2 8 C when not in use Bring reagents to room temperature before use Do not expose reagents to temperatures
7. tion factor to calculate the concentration of human ACE2 in the samples 4 Expected values ACE2 levels range in urine from 1 to gt 10 ng ml from healthy donors Standard hACE2 Optical Density m ng ml mean Technical Hints and Limitations r e It is recommended that all standards QC sample and samples be run in duplicate 2 120 e Do not combine leftover reagents with those reserved for additional wells 12 5 1180 e Reagents from the kit with a volume less than 100 ul should be centrifuged 6 25 0 635 e Residual wash liquid should be drained from the wells after last wash by tapping the PTT rT plate on absorbent paper a e Crystals could appear in the 10X solution due to high salt concentration in the stock K 1903 9 195 solutions Crystals are readily dissolved at room temperature or at 37 C before s 0 701 0 080 dilution of the buffer solutions y 0904 0 092 e Once reagents have been added to the 8 well strips DO NOT let the strips DRY at any time during the assay 14 Y y e Keep TMB Substrate Solution protected from light e The Stop Solution consists of phosphoric acid Although diluted the Stop Solution should be handled with gloves eye protection and protective clothing vi Performance Characteristics Troubleshooting 1 intra assay precision Six samples of known concentrations of human ACE2 were assayed in replicates 11 times to test precision within an assay Omission of key reagent correct order Mean SD
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