Human VWF ELISA Kit
Contents
1. kit is designed for detection of human VWF in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures VWF in less than 5 hours A monoclonal antibody specific for VWF has been pre coated onto a 96 well microplate with removable strips Human VWF in standards and samples is sandwiched by the immobilized antibody and the biotinylated antibody specific for VWF which is recognized by a streptavidin peroxidase conjugate All unbound material is washed away and a peroxidase enzyme substrate is added The color development is stopped and the intensity of the color is measured Caution and Warning e This product is for Research Use Only and is Not For Use In Diagnostic Procedures Prepare all reagents working diluent buffer wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assay e Prepare all samples prior to running the assay The dilution factors for the samples are suggested in this insert However the user should determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e The Stop Solution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Human VWF Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a monoclonal antibody against VWF e Sealing Tapes Each kit co2. 10000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 400 ul or equal to 400 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 4 ul of A 396 ul buffer 100x 1000 fold dilution 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 240 ul or equal to 240 ul Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute MIX Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 64 mU of Human VWF Standard with 0 8 ml of MIX Diluent to generate an 80 mU ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting the VWF standard stock solution 80 mU ml 1 2 with equal volume of MIX Diluent to produce 40 20 10 5 and 2 5 mU ml solutions MIX Diluent serves as the zero standard 0 mU ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point P1 1 part Standard 80 mU ml 1 part P1 1 part M
3. IX Diluent 40 00 1 part P2 1 part MIX Diluent 20 00 Dilution Pa IpartP3 1partMIXDiluent 10 00 Ps 1partPS 1partMiXDient 2 500 e Biotinylated Human VWF Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with MIX Diluent Any remaining solution should be frozen at 20 C e Wash Buffer Concentrate 20x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the Wash Buffer Concentrate 1 20 with reagent grade water e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1 100 with MIX Diluent Any remaining solution should be frozen at 20 C Assay Procedure e Prepare all reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is performed at room temperature 20 25 C e Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator e Add 50 ul of Human VWF Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash five times with 200 ul of Wash Buffer manually Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liqu
4. absorbance OD on the y axis The best fit line can be determined by regression analysis using log log or four parameter logistic curve fit Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data The typical data is provided for reference only Individual laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 80 00 P2 40 00 P3 20 00 P4 10 00 PS 5 000 P6 2 500 P7 0 000 Sample Pool Normal Sodium Citrate Plasma 100x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed vWF Standard Curve OD 450 nm 0 1 po 1 ul AAA 1 10 100 vWF mU ml Reference Value e Normal human plasma VWF concentration has been reported ranging approximately from 0 3 to 1 57 IU ml 4 Normal citrated human plasma VWF values are 0 52 1 54 IU ml for O blood group subjects and 0 6 2 0 IU ml for non O blood group subjects 5 e Human plasma and serum samples from healthy adults were tested n 40 On average VWF level was 1 2 IU ml Sample Average Value IU ml Human Pool Normal Plasma 1 0 Human Normal Plasma 1 2 Human Pool Normal Serum 1 4 Note e The conversion of IU and ug is 1 IU ml 9 8 ug ml e _ The convers
5. e provided procedure for correct incubation prolonged incubation time periods e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples Contamination of e Anew tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the assay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 ZimmermanT S et al 1987 Human Pathology 18 140 2 OkumuraT etal 1976 Thromb Res 8 701 3 Morton L F etal 1983 Thromb Res 32 545 Version 7 9R www assaypro com e e mail Support assaypro com
6. id If using a machine wash six times with 300 ul of Wash Buffer and then invert the plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid Add 50 ul of Biotinylated Human VWF Antibody to each well and incubate for 2 hours Wash the microplate as described above Add 50 ul of Streptavidin Peroxidase Conjugate per well and incubate for 30 minutes Turn on the microplate reader and set up the program in advance Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 20 minutes or till the optimal blue color density develops Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip Add 50 ul of Stop Solution to each well The color will change from blue to yellow Read the absorbance on a microplate reader at a wavelength of 450 nm immediately If wavelength correction is available subtract readings at 570 nm from those at 450 nm to correct optical imperfections Otherwise read the plate at 450 nm only Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes which will reduce the readings Data Analysis Calculate the mean value of the duplicate or triplicate readings for each standard and sample To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm
7. ion of IU and mU is 1 IU ml 1000 mU ml Performance Characteristics e The minimum detectable dose of VWF as calculated by 25D from the mean of a zero standard was established to be 1 1 mU ml e Intra assay precision was determined by testing replicates of three plasma samples in one assay e Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 5 20 mU ml Recovery 86 118 Average Recovery 99 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 50 92 92 1 100 99 98 1 200 Cross Reactivity Species 106 104 Cross Reactivity Canine None Bovine None Monkey lt 40 Mouse None Rat lt 15 Swine None Rabbit Troubleshooting None Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components from different lots e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration Improper wash step e Check that the microplate washer is dispe
8. nsing properly e If washing by pipette check for proper pipetting c technique o Splashing of reagents e Pipette properly in a controlled and careful manner 8 while loading wells gL A e Pipette properly in a controlled and careful manner a Inconsistent volumes X PATA z loaded into wells e Check pipette calibration o e Check pipette for proper performance a Insufficient mixing of Thoroughly agitate the lyophilized components after ED reconstitution reagent dilutions eh e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Microplate was left e Each step of the procedure should be performed 3 unattended between uninterrupted 2 T steps 7 z Omission of step e Consult the provided procedure for complete list of steps oY e Steps performed in e Consult the provided procedure for the correct order E G g incorrect order Q I S insufficient amount of e Check pipette calibration a o dded t i go reagents added to e Check pipette for proper performance wells Wash step was skipped e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent e Consult reagent preparation section for the correct preparation dilutions of all reagents Insufficient or e Consult th
9. ntains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Human VWF Standard Human VWF in a buffered protein base 64 mU lyophilized calibrated against WHO 1 International Standard e Biotinylated Human VWF Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against VWF 120 pul e MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles e Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul e Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition e Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date e Store SP Conjugate and Biotinylated Antibody at 20 C e Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C e Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 daysina vacuum desiccator e Diluent 1x may be stored for up to 30 days at 2 8 C e Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent O
10. ther Supplies Required e Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 100 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA or Heparin can also be used as an anticoagulant e Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes and remove serum Dilute samples 1 100 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay The samples can be stored at 20 C or below Avoid repeated freeze thaw cycles Refer to Sample Dilution Guidelines below for further instruction Guidelines for Dilutions of 1 100 or Greater for reference only please follow the insert for specific dilution suggested 1 100 1 10000 4 ul sample 396 ul buffer 100x A 4ulsample 396 ul buffer 100x 100 fold dilution 4 ul of A 396 ul buffer 100x
11. yssarpro AssayMax Human VWF ELISA Kit Assaypro LLC 3400 Harry S Truman Blvd St Charles MO 63301 T 636 447 9175 F 636 395 7419 WWW assaypro com For any questions regarding troubleshooting or performing the assay please contact our support team at support assaypro com Thank You for choosing Assaypro Assay Summary Step 1 Add 50 ul of Standard or Sample per well Incubate 2 hours Step 2 Wash then add 50 ul of Biotinylated Antibody per well Incubate 2 hours Step 3 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 4 Wash then add 50 ul of Chromogen Substrate per well Incubate 20 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key ci Consult instructions for use Assay Template 12 11 10 Human Von Willebrand Factor VWF ELISA Kit Catalog No EV2030 1 Sample insert for reference use only Introduction Von Willebrand factor VWF is a multimeric glycoprotein that circulates in blood forming a noncovalent complex with procoagulant factor VIII 1 During normal homeostasis the larger multimers of VWF are responsible for facilitating platelet plug formation by forming a bridge between platelet glycoprotein IB and exposed collagen in the subendothelium 2 3 Principle of the Assay The AssayMax VWF ELISA Enzyme Linked Immunosorbent Assay
Download Pdf Manuals
Related Search