Human IFN-gamma ELISA Kit
Contents
1. kDa 4 By binding to the receptors IFNGR1 amp IFNGR2 IFN gamma activates the tyrosine kinase JAK STAT pathway 5 While protecting against tumor development and cancer immunoediting IFN gamma function is significant in tumor surveillance 6 Aside from functions in host defense IFN gamma may contribute to autoimmune pathology 7 10 Principle of the Assay The AssayMax Human Interferon gamma ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human IFN gamma in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human IFN gamma in less than 5 hours A polyclonal antibody specific for human IFN gamma has been pre coated onto a 96 well microplate with removable strips IFN gamma in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for IFN gamma 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 fac2. A assarbno AssayMax Human IFN gamma 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 25 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 Interferon gamma ELISA Kit Catalog No El1023 1 Sample insert for reference use only Introduction Interferon gamma IFN gamma is a highly pleiotropic protein secreted mainly by activated T lymphocytes and natural killer cells It is involved in a wide range of physiological processes including antiviral immunoregulatory and anti tumour properties cell proliferation and apoptosis as well as the stimulation and repression of a variety of genes 1 3 IFN gamma is a homodimer consisting of two 143 amino acid polypeptides with 20 kDa and 25
3. en 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 Human Interferon y Standard Curve 1 0r OD 450 nm P 1 10 107 10 Interferon y ng ml Reference Value e Human plasma and serum samples from healthy adults were tested n 20 On average IFN gamma level was 39 pg ml However some samples measured less than the lowest standard 15 6 pg ml Performance Characteristics e The minimum detectable dose of IFN gamma as calculated by 2SD 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 CV 96 Average CV 96 Recovery Standard Added Value 0 031 0 5 ng ml Recovery 96 94 10996 Average Recovery 96 9796 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum No Dilution 10096 9996 1 2 9996 102 1 4 97 97 Cross Reactivity Species Cross Reactiv
4. ity Beagle None Bovine None Monkey None Mouse None Rat None Rabbit None Swine 80 Human 100 Troubleshooting 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 Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions c 2 2 o o a 3 o Inconsistent volumes loaded into wells Insufficient mixing of reagent 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 li
5. jugate 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 and assay 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 assay 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 Store samples at 20 C or below Avoid repeated freeze tha
6. oper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions References 1 2 3 4 5 6 7 8 9 Boehm U etal 1997 Annu Rev Immunol 15 749 795 Schoenborn JR and Wilson CB 2007 Adv Immunol 96 41 101 Schroder K et al 2004 J Leukoc Biol 75 163 189 Rinderknecht E et al 1984 J Biol Chem 259 6790 6797 Kaplan DH et al 1996 J Biol Chem 271 9 12 Ikeda H et al 2002 Cytokine Growth Factor Rev 13 95 109 Baechler EC et al 2003 Proc Natl Acad Sci USA 100 2610 2615 Panitch HS et al 1987 Lancet 1 893 895 Sarvetnick N et al 1988 Cell 52 773 782 10 Key LLet al 1992 J Pediatr 121 119 124 Version 1 8R www assaypro com e mail Support assaypro com
7. st 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 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 pr
8. 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 25 minutes or till the optimal blue color density develops Gently tap 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 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 betwe
9. tors 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 Thekit should not be used beyond the expiration date Reagents e Human IFN gamma Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human IFN gamma 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 IFN gamma Standard Human IFN gamma in a buffered protein base 2 ng lyophilized e Biotinylated Human IFN gamma Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against IFN gamma 120 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 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 Con
10. w cycles Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 2 ng of Human IFN gamma Standard with 2 ml of EIA 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 ng ml 1 2 with EIA Diluent to produce 0 5 0 25 0 125 0 063 0 031 and 0 016 ng ml solutions EIA 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 IFN gamma ng ml P1 1 part Standard 1 ng ml 1 0000 1 part P1 1 part EIA Diluent 0 5000 1 part P2 1 part EIA Diluent 0 2500 P4 1partP3 1 part EIA Diluent 0 1250 PS ipartPS ipartElADiluent 0 0313 Ll P8 EADiuent 0000 e Biotinylated Human IFN gamma Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 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 gentl
11. y 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 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 IFN gamma 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 Human IFN gamma Antibody to each well and incubate for 2 hours e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate to each well and incubate for 30 minutes Turn on
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