AssayMaxTM Human Factor V ELISA Kit
Contents
1. Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 2 with EIA 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 Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 2 with EIA 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 CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 2 into EIA Diluent and assay The undiluted samples can be stored at 80 C for up to 3 months 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 protocol for specific dilution suggested 1 100 1 10000 A 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is less than or equal to 400 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than or equal to 400 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 1000 fold dilution Assuming the needed volume is less than or equal to 240 ul 4 ul sam2. Wash the microplate as described above e 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 e Wash the microplate as described above e 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 e Add 50 ul of Stop Solution to each well The color will change from blue to yellow e 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 e Calculate the mean value of the duplicate or triplicate readings for each standard and sample e To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm 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 e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution facto
3. the presence of a calcium ion and the phospholipid on cell surfaces FVa and FXa form the prothrombinase complex which catalyzes the hydrolysis of prothrombin to thrombin 3 Thrombin in turn cleaves fibrinogen to fibrin which polymerizes to form a clot FVa is readily inactivated by anticoagulant activated protein C 4 FV Leiden a single amino acid mutation renders FVa resistant to cleavage by activated protein C It therefore over produces thrombin and leads to excess clotting and hereditary thrombophilia disorder 5 Severe FV deficiency is associated with mild to severe bleeding diathesis 6 Principle of the Assay The AssayMax Human Factor V ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human factor V in plasma urine milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures factor V in less than 4 hours A monoclonal antibody specific for factor V has been pre coated onto a 96 well microplate with removable strips Factor V in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for factor V 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 Prepare all reagents working diluent buffer wash buffe
4. MA assarbro AssayMax Human Factor V 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 1 hour 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 BE Consult instructions for use Assay Template 12 11 10 Human Factor V ELISA Kit Catalog No EF1005 1 Sample protocol for reference use only Introduction Factor V FV is an essential cofactor of the blood coagulation cascade and circulates in plasma as a large single chain glycoprotein The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28 amino acid leader peptide 1 During coagulation it is converted to the active cofactor FVa via limited proteolysis by thrombin and spliced into a heavy chain 110 KDa and a light chain 73 KDa held together non covalently by calcium 2 In
5. d 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 days in a 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 Other 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 1 800 with EIA 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 Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store the remaining samples at 20 C or below
6. ng two plasma samples with different factor V concentrations Unspik se Spike Recovery Sample Sample ml Expected Observed 9 ng ml inem 2 5 7 7 8 1 105 1 5 2 5 0 10 2 10 3 101 10 0 15 2 14 9 98 2 5 4 9 5 4 110 2 2 4 5 0 7 4 7 5 101 10 0 12 4 11 9 96 Average Recovery 102 Linearity e Plasma samples were serially diluted to test for linearity Average Percentage of Expected Value 96 Sample Dilution Plasma 1 400 94 1 800 98 1 1600 Cross Reactivity 105 Species Cross Reactivity Canine None Bovine None Monkey lt 15 Mouse None Rat None Swine lt 15 Rabbit None Troubleshooting Issue Causes Course of Action Use of expired e Check the expiration date listed before use c components e Do not interchange components from different lots 2 e Check that the correct wash buffer is being used D e Check that all wells are dry after aspiration E Improper wash step e Check that the microplate washer is dispensing properly z e If washing by pipette check for proper pipetting o technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mi
7. ple 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than or equal to 240 ul Reagent Preparation Standard Point Freshly dilute all reagents and bring all reagents to room temperature before use EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C Standard Curve Reconstitute the 210 ng of Human Factor V Standard with 3 5 ml of EIA Diluent to generate a 60 ng 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 standard stock solution 60 ng ml 1 2 with EIA Diluent to produce 30 15 7 5 3 75 1 875 and 0 938 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution Dilution should be frozen at 20 C and used within 30 days FV ng ml 60 00 P1 1 part Standard 60 ng ml 1 part P1 1 part EIA Diluent 30 00 1 part P2 1 part EIA Diluent 15 00 P4 A part P3 1 part EIA Diluent 7 500 Ps ipatP5 lparttlADiuent 1875 Ll P8 EA Diluent 000 e Biotinylated Human Factor V Antibody 60x Spin down the antibody briefly and dilute the desired amount of the antibod
8. r Typical Data e 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 60 00 ES 1 836 P2 30 00 X 1 503 P3 15 00 us 1 131 P4 7 500 ise 0 795 P5 3 750 MM 0 574 P6 1 875 ee 0 495 P7 0 938 ae 0 373 P8 0 000 Men 0 211 Sample Pool Normal 0 894 0 896 Sodium Citrate Plasma 800x 0 898 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed hFV Standard Curve OD 450 nm 0 1 L ey per op A 1 1 0 10 0 100 0 hFV ng ml Reference Values e Normal human factor V plasma levels range from 4 to 16 ug ml e Human plasma samples from healthy adults were tested n 20 On average factor V level was 8 8 ug ml Performance Characteristics e The minimum detectable dose of factor V as calculated by 2SD from the mean of a zero standard was established to be 0 9 ng ml 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 Spiking Recovery e Recovery was determined by spiki
9. r 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 protocol 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 This kit is for research use only e The kit should not be used beyond the expiration date Reagents e Human Factor V Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a monoclonal antibody against factor V e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Human Factor V Standard Human factor V in a buffered protein base 210 ng lyophilized e Biotinylated Human Factor V Antibody 60x An 60 fold concentrated biotinylated polyclonal antibody against human factor V 100 ul e EIA 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 aci
10. values lower than the highest standard point P1 dilute samples further and repeat the assay User should determine the optimal dilution factor for samples Contamination of e A new 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 Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions 10 References 1 2 3 4 5 6 Jenny RJ et al 1987 Proc Natl Acad Sci U S A 84 4846 4850 Esmon CT 1979 J Biol Chem 254 964 973 Nesheim ME etal 1981 J Biol Chem 256 6537 6540 Kisiel W et al 1977 Biochemistry 16 5824 5831 Bertina RM et al 1994 Nature 369 64 67 Asselta R et al 2006 J Thromb Haemost 4 1 26 34 Version 1 5 www assaypro com e e mail Support assaypro com
11. xing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Improperly sealed microplate e Check the microplate pouch for proper sealing e Check that the microplate pouch has no punctures e Check that three desiccants are inside the microplate pouch prior to sealing Unexpectedly Low or High Signal Intensity Microplate was left unattended between steps e Each step of the procedure should be performed uninterrupted Omission of step e Consult the provided procedure for complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance 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 preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD
12. y 1 60 with EIA 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 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 EIA 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 Factor V Standard or sample per well Cover wells 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 liquid 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 e Add 50 ul of Biotinylated Human Factor V Antibody to each well and incubate for 1 hour e
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