AssayMax Mouse TAT Complex ELISA Kit
Contents
1. 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 0 1 2 ng ml Recovery 89 115 Average Recovery 96 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma 1 2 86 1 4 97 1 8 Cross Reactivity Species 104 Cross Reactivity Beagle None Bovine None Monkey 10 Rat 10 Swine 10 Rabbit None Human None Mouse Troubleshooting 100 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 dispensing properly e If washing by pipette check for proper pipetting technique o Splashing of reagents e Pipette properly in a controlled and careful manner 8 while loading wells y y e Pipette properly in a controlled and careful manner a Inconsistent volumes y cd e Check pipette calibration 3 loaded into wells 7 3 e Check pipette for proper performance Insuff2. by serially diluting the standard stock solution 1 4 with MIX Diluent to produce 2 0 5 0 125 and 0 031 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 7 days Standard Point Dilution Mouse TAT ng ml P1 1 part Standard 8 ng ml 8 0000 1 part P1 3 parts MIX Diluent 2 0000 1 part P2 3 parts MIX Diluent 0 5000 P4 1patP3 3 parts MIX Diluent 0 1250 1 part P4 3 parts MIX Diluent 0 0313 PSS MIX Diluent 0 0000 e Biotinylated Mouse Antithrombin 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 expo
3. es This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse TAT complex in less than 5 hours A polyclonal antibody specific for mouse thrombin has been pre coated onto a 96 well microplate with removable strips TAT complex in standards and samples are sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for mouse antithrombin which is recognized by a streptavidin peroxidase conjugate All unbound material is then 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 Mouse TAT Complex Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against mouse thrombin e Sealing Tapes Each kit contains 3 p
4. icient mixing of Thoroughly agitate the lyophilized components after a reconstitution reagent dilutions IRA ye 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 gt gt unattended between uninterrupted gt H a steps 2 I 9 Omission of step e Consult the provided procedure for complete list of steps 2 Steps performed in e Consult the provided procedure for the correct order 5350 incorrect order Q m Insufficient amount of e Check pipette calibration n reagents added to 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 the 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 furthe
5. ies 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 mouse plasma 1 4 with 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 Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Samples can be stored at 20 C or below for up to 30 days Avoid repeated freeze thaw cycles 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 the MIX Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Mouse TAT Complex Standard Reconstitute the 4 ng of Mouse TAT Complex Standard with 0 5 ml of MIX Diluent to generate an 8 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
6. r and repeat the assay e 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 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 Diquelou A et al 1994 Blood 84 7 2206 13 2 Heller MV etal 1995 Thromb Haemost 73 3 368 73 Version 3 2R Related Product e _ ET1020 1 AssayMax Human Thrombin Antithrombin Complex ELISA Kit Plasma Milk and Cell Culture samples www assaypro com e mail Support assaypro com
7. recut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Mouse TAT Complex Standard Mouse TAT complex in a buffered protein base 4 ng lyophilized 2 vials e Biotinylated Mouse Antithrombin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against mouse antithrombin 140 ul 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 Other Suppl
8. sure to water vapor and store in a vacuum desiccator e Add 50 ul of Mouse TAT Complex 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 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 Mouse Antithrombin Antibody to each well and incubate for 2 hours e 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 15 minutes or till the optimal 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 no
9. te 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 on the y axis The best fit line can be determined by regression analysis using four parameter or log log logistic curve fit e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor 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 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Mouse TAT Complexes Standard Curve OD 450 nm 0 01 0 10 1 00 10 00 100 00 TAT ng ml Performance Characteristics e The minimum detectable dose of mouse TAT as calculated by 25D from the mean of a zero standard was established to be 0 01 ng 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
10. yssaypro AssayMax Mouse TAT Complex 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 15 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 Mouse TAT Complex ELISA Kit Catalog No EMT1020 1 Sample insert for reference use only Introduction Thrombin antithrombin TAT complex formed following the neutralization of thrombin by antithrombin III ATIII have been used as a surrogate marker for thrombin generation 1 High plasma levels of TAT complexes have been suggested to alter hemostatic activation 2 Principle of the Assay The AssayMax Mouse TAT Complex ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of mouse TAT complex in plasma and cell culture sampl
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