Rat Tumor Necrosis Factor-alpha (TNF-alpha) ELISA Kit
Contents
1. CV Average CV Troubleshooting Causes Course of Action Low Precision Use of expired components e Check the expiration date listed before use e Do not interchange components from different lots Improper wash step e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner 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 mixing 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 c o N lt e o wn 2 so Ze mo w 2 oO w Q x c gt 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 Steps2. The AssayMax Rat Tumor Necrosis Factor alpha ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of TNF alpha in rat cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures rat TNF alpha in less than 5 hours A murine monoclonal antibody specific for rat TNF alpha has been pre coated onto a 96 well microplate with removable strips Rat TNF alpha in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for rat TNF alpha 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 The kit should not be used beyond the expiration date Reagents Rat TNF alpha Microplate A 96 wel
3. 2 Dilution Point ng ml P1 1 part Standard 1 ng ml 1 000 1 part P1 1 part MIX Diluent 0 500 1 part P2 1 part MIX Diluent 0 250 Pa 1ipartP3 1partMiXDiluent 0 125 Pe 1partP5 1 part MIX Diluent 0031 Ps MiXDiluent 0o00 e Biotinylated Rat TNF alpha 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 Rat TNF alpha 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
4. 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 values lower 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 reagents e A new tip must be used for each addition of different samples or reagents during the assay procedure Contents of wells evaporate e Verify that the sealing film is firmly in place before placing 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 performa
5. reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel Deionized or distilled reagent grade water Sample Collection Preparation and Storage 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 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 Standard Curve Reconstitute the 1 5 ng of Rat TNF alpha Standard with 1 5 ml of MIX Diluent to generate a 1 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 1 2 with equal volume of MIX Diluent to produce 0 5 0 25 0 125 0 063 0 031 and 0 016 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and use within 30 days Standard are Rat TNF alpha
6. 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 Rat TNF alpha 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 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 a
7. l polystyrene microplate 12 strips of 8 wells coated with a murine monoclonal antibody against rat TNF alpha Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Rat TNF alpha Standard Rat TNF alpha in a buffered protein base 1 5 ng lyophilized Biotinylated Rat TNF alpha Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against rat TNF alpha 120 ul MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store SP Conjugate and Biotinylated Antibody at 20 C Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C 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 Diluent 1x may be stored for up to 30 days at 2 8 C Store Standard at 2 8 C before
8. nce e Thoroughly agitate the lyophilized components after Insufficient mixing of rn Ing reconstitution reagent dilutions e Thoroughly mix dilutions References 1 Taylor PC 2001 Mol Biotechnol 19 2 153 68 2 Coughlan MT et al 2001 Diabet Med 18 11 921 7 3 Stamm C et al 2001 Circulation 104 12 Suppl 1 1350 5 Version 3 3R Related products ET2010 1 AssayMax Human TNF alpha ELISA Kit Plasma Serum and Cell Culture samples EMT2010 1 AssayMax Mouse TNF alpha ELISA Kit Plasma Serum and Cell Culture samples www assaypro com e mail Support assaypro com
9. nd 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 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 R TNF alpha Standard Curve OD 450 nm 0 01 zu 510 1 00 rTNF alpha ng ml Performance Characteristics e This assay recognizes both natural and recombinant rat TNF alpha e This kit can be used to detect high TNF alpha levels in rat plasma e The minimum detectable dose of rat TNF alpha 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 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
10. yssarpro AssayMax Rat TNF alpha 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 Rat Tumor Necrosis Factor alpha TNF alpha ELISA Kit Catalog No ERT2010 1 Sample insert for reference use only Introduction Tumor necrosis factor alpha TNF alpha is a potent cytokine with a myriad of innate immune anti tumor properties TNF alpha has a critical role in the bone and cartilage damage 1 Research indicates that TNF alpha may be involved in the pathogenesis and or progression of gestational diabetes mellitus GDM 2 TNF alpha is expressed in myocardium during compensated pressure overload hypertrophy 3 Principle ofthe Assay
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