ANGPTL3 (mouse/rat) Serum ELISA Kit
Contents
1. Mean SD Recovery When samples serum are spiked with known concentrations of mouse and rat ANGPTL3 the recovery averages 100 range from 90 to 110 Samples Average Recovery Range 93 85 90 100 98 63 95 105 109 56 105 110 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA PROBLEM POSSIBLE CAUSES rev 1 15 For research use only 4 Expected values ANGPTL3 levels range in mouse samples from 50 to gt 1 000 ng ml ANGPTLS levels range in rat samples from 10 to gt 150 ng ml Technical Hints and Limitations e It is recommended that all standards QC sample and samples be run in duplicate e Do not combine leftover reagents with those reserved for additional wells e Reagents from the kit with a volume less than 100 ul should be centrifuged e Residual wash liquid should be drained from the wells after last wash by tapping the plate on absorbent paper e Crystals could appear in the 10X solution due to high salt concentration in the stock solutions Crystals are readily dissolved at room temperature or at 37 C before dilution of the buffer solutions e Once reagents have been added to the 8 well strips DO NOT let the strips DRY at any time during the assay e Keep TMB Substrate Solution protected from light e The Stop Solution consists of sulfuric acid Although diluted the Stop Solution should be handled with gloves eye protection and protective clothing Troubl2. and stored at 20 C Prepare Standard Curve using 2 fold serial dilutions with 1X ELISA Buffer Toobtain Add 300 ul of ANGPTL3 2 ng ml 300 ul of 1X ELISA Buffer 0 5 ng ml 300 ul of ANGPTL3 1 ng ml 300 ul of 1X ELISA Buffer For research use only 0 25 ng ml 300 ul of ANGPTL3 0 5 ng ml 300 ul of 1X ELISA Buffer 0 125 ng ml 300 ul of ANGPTL3 0 25 ng ml 300 ul of 1X ELISA Buffer 0 063 ng ml 300 ul of ANGPTL3 0 125 ng ml 300 ul of 1X ELISA Buffer 0 031 ng ml 300 ul of ANGPTL3 0 063 ng ml 300 ul of 1X ELISA Buffer 0 016 ng ml 300 ul of ANGPTL3 0 031 ng ml 300 ul of 1X ELISA Buffer 0 ng ml 300 ul of Diluent 1X Empty tube 300 ul 300 l 300pul 300ul S 300 300l 300 pI gt bs tE 2 1 0 5 0 25 0 125 0 063 0 031 0 016 0 ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml ng ml 2 Reagent Preparation Prepare just the appropriate amounts for the assay a b C d 1X Wash Buffer Dilute 10X Wash Buffer 1 9 with dH2O to obtain 1X Wash Buffer 1X ELISA Buffer Dilute 10X ELISA Buffer 1 9 with dH2O to obtain 1X ELISA Buffer Detection Antibody has to be diluted to 1 500 in ELISA Buffer 1x Note The diluted Detection Antibody is not stable and cannot be stored HRP Labeled Streptavidin has to be reconstituted with 200 ul of ELISA Buffer 1X prepare aliquots and store them at 20 C Avoid freeze thaw cycles Dilute the reconstituted STREP HRP to the working concentration by adding 50 ul in 10 ml of ELISA Buff
3. for binding to the coated antibody After extensive washing to remove unbound compounds ANGPTLS3 is recognized by the addition of a biotinylated polyclonal antibody specific for ANGPTL3 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 ANGPTL3 in the samples The assay range is 0 016 1 ng ml ANGPTL3 ml The lowest level of ANGPTL3 that can be detected by this assay is 15 pg ml Kit Contents Component 1 plate coated with mouse ANGPTL3 Antibody 2 bottle Wash Buffer 10X 2 bottle ELISA Buffer 10X 1 bottle Detection Antibody 1 vial Detector HRP Labeled Streptavidin 2 ug 1 vial mouse ANGPTL3 Standard lyophilized 2ng 1 bottle TMB Substrate Solution 12 ml K4915 100 7 1 bottle Stop Solution 12 ml K4915 100 8 2 plate sealers plastic film 2 K4915 100 9 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 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 Serum Use a serum separator tube Let samples clot at
4. room temperature for 30 min before centrifugation for 20 min at 1000 x g Assay freshly prepared serum or store serum in aliquots at 20 C for future use Avoid repeated freeze thaw cycles b Plasma Collect using heparin EDTA or citrate as an anticoagulant Centrifugation for 15 min at 1000 x g within 30 min of collection Assay freshly prepared plasma or store in aliquots at 20 C for future use Avoid repeated freeze thaw cycles Note Serum Plasma Urine or Cell Culture Supernatant has to be diluted in Diluent 1X Samples containing visible precipitates must be clarified before use As a starting point 1 4000 dilution of mouse samples and 1 400 dilution of rat samples are recommended c QC Sample Reconstitute Mouse ANGPTL3 QC sample with 1 ml of dH2O 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 100 Assays Part Number 6x16 well strips K4915 100 1 2x30 ml K4915 100 2 2x30 ml K4915 100 3 30 ul K4915 100 4 1 vial K4915 100 5 1 vial K4915 100 6 BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA d e rev 1 15 Standards Reconstitute mouse ANGPTL3 Standard with 1 ml of dH2O to produce a stock solution 2 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
5. BioVision ANGPTL3 mouse rat Serum ELISA Kit Catalog K4915 100 100 assays Store kit at 4 C Description The angiopoietins are a family of growth factors that are specific for vascular endothelium The full length cDNA encoding angiopoietin like protein 3 ANGPTL3 from a human fetal liver spleen cDNA library has 460 amino acid and the characteristic structure of angiopoietins a signal peptide an extended helical domain predicted to form dimeric or trimeric coiled coils a short linker peptide and a globular fibrinogen like domain FLD Human ANGPTL3 shares 76 amino acid sequence identity with mouse Angptl3 Northern blot analysis of human tissues showed a preferential expression of 4 ANGPTLS transcripts being 4 5 3 0 2 8 and 1 7 kb in liver ANGPTL3 can induce angiogenesis in the rat corneal assay The FLD alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis Microarray analysis showed that mouse hematopoietic stem cell HSC supportive fetal liver CD3 positive cells expressed Angptl2 and Angptl3 The ANGPTL3 ELISA Kit is to be used for the in vitro quantitative determination of mouse rat ANGPTL3 in biological fluids This assay is a sandwich Enzyme Linked Immunosorbent Assay ELISA for quantitative determination of mouse or rat ANGPTL3 in biological fluids A polyclonal antibody specific for ANGPTL3 has been precoated onto the 96 well microtiter plate Standards and samples are pipetted into the wells
6. er 1X 1 200 Note The diluted STREP HRP is not stable and cannot be stored Assay Protocol Determine the number of 16 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 and Samples into the appropriate wells in duplicate Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash 3 times with 300 ul of 1X Wash Buffer Completely remove the liquid Add 100 ul detection antibody into each well Cover plate with plate sealer and incubate for 1 hr at 37 C Aspirate and wash 3 times with 300 ul of 1X Wash Buffer Completely remove the liquid Add 100 ul of diluted STREP HRP into each well Cover plate with plate sealer and incubate for 1 hr at 37 C Remove plate from 37 C aspirate and wash 5 times 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 to each well of TMB Substrate Solution Allow the color to develop at room temperature in the dark for 10 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 Fax 408 493 1801 tech biovision com Tel 408 493 1800 www biovision com Page 1 of 2 BioVision solution that turns yellow when St
7. eshooting SOLUTIONS ce Check that all reagents have been added in the Washes too stringent Use an automated plate washer if possible Nasiona k Incubation times Incubation times should be followed as pee as or Weak inadequate indicated in the manual g late reader settings not Verify the wavelength and filter setting in the optimal plate reader Incorrect assay temperature Use recommended incubation temperature Bring substrates to room temperature before use Concentration of 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 aspirated Completely aspirate wells between steps Poor standard E Reagents poorly mixed Be sure that reagents are thoroughly mixed PE Be sure that reagents were prepared correctly Unexpected OMISSION OrEAJENS and added in the correct order results ipetti i z Dil tion error Check pipetting technique and double check calculations FOR RESEARCH USE ONLY Not to be used on humans Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 2
8. op Solution is added Caution Stop Solution is a Corrosive Solution p Measure the OD at 450 nm in an ELISA plate reader within 30 min Calculations a Average the duplicate readings for each Standard QC Sample and Test Sample and subtract the average blank value obtained with the 0 ng ml point b Generate a Standard Curve by plotting the average absorbance on the horizontal X axis vs the corresponding concentration ug ml on the vertical Y axis See Typical Data below c Calculate the Test Sample ANGPTL3 concentrations by interpolation of the Standard Curve regression curve as shown below in the form of a quadratic equation d Ifthe Test Samples were diluted multiply the interpolated values by the dilution factor to calculate the corrected mouse or rat ANGPTL3 concentrations 1 2 2 6 38 ee gue Standard Optical Density 1 is gt mANGPTL3 ng ml mean ps 1 2 128 E 0 5 1 216 06 A 0 25 0 570 0 4 a 0 125 0 268 02 P i 0 0625 0 113 M 0 0312 0 068 oO T 0 000 0 500 1 000 1 500 2 000 2 500 0 0156 0 009 OD o 0 000 Figure Standard curve Performance Characteristics Intra assay precision Six samples of known concentrations of mouse and rat ANGPTL3 were assayed in replicates 10 times to test precision within an assay Mean 512 29 Inter assay precision Five samples of known concentrations of mouse and rat ANGPTL3 were assayed in 5 separate assays to test precision between assays 6
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