AssayMaxTM Human Transferrin ELISA Kit
Contents
1. 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 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 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 mann2. 06 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 5 Mouse None Rat None Swine None Human 100 Troubleshooting 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 Low Precision 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 Unexpectedly Low or High Signal Intensity Microplate was left unattended between steps e Each step of the procedure should be performed uninterrupted Omission of
3. ces Standard Point Average OD P1 25 00 P2 6 250 P3 1 563 P4 0 391 P5 0 098 P6 0 000 Sample Pool Normal Sodium Citrate Plasma 2000x Standard Curve The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Transferrin Standard Curve 10 00 1 00 OD4s0 nm 0 10 0 01 i en 0 1 1 0 10 0 100 0 Transferrin ug ml Reference Value e Normal human transferrin plasma levels range from 2 to 4 mg ml Human plasma and serum samples from healthy adults were tested n 40 On average transferrin level was 3 09 mg ml Performance Characteristics The minimum detectable dose of transferrin as calculated by 2SD from the mean of a zero standard was established to be 0 07 ug ml Intra assay precision was determined by testing replicates of three plasma samples in one assay 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 0 3 10 ug ml Recovery 88 111 Average Recovery 96 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 1000 101 93 1 2000 99 97 1 4000 104 1
4. concentrated buffered protein base 30 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 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 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 and Biotinylated Protein 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 2000 int
5. e e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 10 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 low 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 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 differen
6. er 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 Reference 1 Averbukh Zet al 2004 J Nephrol 17 1 101 6 Version 5 3R Related Products ET3105 1 AssayMax Human Transferrin ELISA Kit Urine Milk Saliva and Cell Culture samples e ERT2105 1 AssayMax Rat Transferrin ELISA Kit Plasma and Serum samples e ERT3105 1 AssayMax Rat Transferrin ELISA Kit Urine and Cell Culture samples EMT2105 1 AssayMax Mouse Transferrin ELISA Kit Plasma Serum and Cell Culture samples www assaypro com e e mail Support assaypro com
7. ll microplate with removable strips Transferrin in standards and samples is competed with a biotinylated transferrin sandwiched by the immobilized antibody and 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 protein 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 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 Transferrin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human transferrin 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 Transferrin Standard Human transferrin in a buffered protein base 75 ug lyophilized e Biotinylated Human Transferrin 1 vial lyophilized e _ MIX Diluent Concentrate 10x A 10 fold
8. o 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 2000 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 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 4 ul sample 396 ul buffer 100x 100 fold dilution 4 ul of A 396 ul buffer 100x 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 C
9. oncentrate 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 75 ug of Human Transferrin Standard with 3 ml of MIX Diluent to generate a 25 ug 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 25 ug ml 1 4 with MIX Diluent to produce 6 25 1 563 0 391 and 0 098 ug ml solutions MIX Diluent serves as the zero standard 0 pg ml Any remaining solution should be frozen at 20 C and used within 30 days Standard i a Transferrin 3 Dilution Point ug ml P1 1 part Standard 25 ug ml 25 00 1 part P1 3 parts MIX Diluent 6 250 Pa 1partP3 3parsMIXDiluent 0301 Ps MiXDiluent 0o00 e Biotinylated Human Transferrin 6x Reconstitute Biotinylated Human Transferrin with 4 ml MIX Diluent to produce a 6 fold stock solution Allow to sit for 10 minutes with gentle agitation prior to making dilutions The stock solution should be further diluted 1 6 with MIX Diluent Any remaining solution should be frozen at 20 C and used within 30 days e Wash Buffer Concentrate 20x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dil
10. ute 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 25 ul of Human Transferrin Standard or sample per well and immediately add 25 ul of Biotinylated Human Transferrin to each well on top of the standard or sample and tap plate to mix gently Cover wells with a sealing tape and incubate for 1 hour 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 Streptavidin Peroxidase Conjugate to each well and incubate for 30 minutes Turn on the microplate reader and set up the program in advanc
11. yssarpro AssayMax Human Transferrin 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 25 ul of Standard or Sample and 25 ul of Biotinylated Protein per well Incubate 1 hour Step 2 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 3 Wash then add 50 ul of Chromogen Substrate per well Incubate 10 minutes Step 4 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 Transferrin ELISA Kit Catalog No ET2105 1 Sample insert for reference use only Introduction Transferrin is a plasma protein that transports iron through the blood to the liver spleen and bone marrow Principle of the Assay The AssayMax Human Transferrin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human transferrin in plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures human transferrin in less than 2 hours A polyclonal antibody specific for human transferrin has been pre coated onto a 96 we
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