AssayMax Rat Ceruloplasmin ELISA Kit
Contents
1. 20 20 20 CV 96 Average CV 96 Recovery Standard Added Value 1 10 ng ml Recovery 96 82 113 Average Recovery 96 95 Linearity e Urine samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Urine No Dilution 89 1 2 99 1 4 Cross Reactivity Species 106 Cross Reactivity Beagle None Bovine None Monkey None Mouse lt 5 Human None Swine Troubleshooting lt 2 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 o e Check that all wells are dry after aspiration 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 mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Imp2. 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 Ceruloplasmin Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last sample 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 Rat Ceruloplasmin Antibody to each well and incubate for 1 hour 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 10 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 fro
3. region of the brain 1 3 5 Principle of the Assay The AssayMax Rat Ceruloplasmin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of rat ceruloplasmin in urine and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures rat ceruloplasmin in less than 4 hours A polyclonal antibody specific for rat ceruloplasmin has been pre coated onto a 96 well microplate with removable strips Ceruloplasmin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ceruloplasmin 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 expi
4. A assarbno AssayMax Rat Ceruloplasmin 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 10 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 Ceruloplasmin ELISA Kit Catalog No ERC4101 1 Sample insert for reference use only Introduction Ceruloplasmin is an abundant a2 serum glycoprotein that contains 9596 of the copper found in the plasma of vertebrate species 1 Ceruloplasmin is a copper binding protein that normally removes iron from cells by its ferroxidase activity On average ceruloplasmin concentration is 14 6 4 0 mg dl 2 Low levels of ceruloplasmin lead to the abnormal deposition of iron in cells including those of the pancreas liver retina and the basal ganglia
5. aking dilutions Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 20 ng ml 1 2 with equal volume of MIX Diluent to produce 10 5 2 5 1 25 0 625 and 0 313 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 30 days Standard Rat Ceruloplasmin Point ng ml 1 part Standard 20 ng ml 20 00 1 part P1 1 part MIX Diluent 10 00 1 part P2 1 part MIX Diluent 5 000 Pa 1partP3 1partMiXDiluent 250 Pe tipatPS ipartMiXDiuent 065 P8 MixDiluent 0000 e Biotinylated Rat Ceruloplasmin Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 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
6. cator 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 e Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel pipette e Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 2 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 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 RatCeruloplasmin Standard Reconstitute the 40 ng of Rat Ceruloplasmin Standard with 2 ml of MIX Diluent to generate a 20 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to m
7. m 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 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Rat Ceruloplasmin Standard Curve 2 T OD 450nm 0 1 1 1 J 1 0 1 2 Ceruloplasmin ng ml Performance Characteristics e The minimum detectable dose of rat ceruloplasmin as calculated by 2SD from the mean of a zero standard was established to be 0 2 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
8. ne 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 References 1 2 3 4 5 e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Harris Z L et al Proc Natl Acad Sci Vol 92 pp 2539 2543 March 1995 Aliyazicioglu Y et al The Turkish Journal of Pediatics 2007 49 52 54 Czaja M J et al J Clin Invest Volume 80 October 1987 1200 1204 Kumar A et al WJM October 1995 Vol 163 No 4 Moller L B et al Am J Hum Genet 66 1211 1220 2000 Version 1 3R3 Related Products EC4101 1 AssayMax Human Ceruloplasmin ELISA Kit Urine Saliva Milk and Cell Culture samples e EC4001 1 AssayMax Human Ceruloplasmin ELISA Kit Plasma and Serum samples e ERC4001 1 AssayMax Rat Ceruloplasmin ELISA Kit Plasma and Serum samples www assaypro com e e mail Support assaypro com
9. ration date Reagents e RatCeruloplasmin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against rat ceruloplasmin 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 RatCeruloplasmin Standard Rat Ceruloplasmin in a buffered protein base 40 ng lyophilized e Biotinylated Rat Ceruloplasmin Antibody 100x A 100 fold biotinylated polyclonal antibody against rat ceruloplasmin 80 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 days in a vacuum desic
10. roperly 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 values lower than the highest standard point P1 dilute samples further and repeat the assay User should determi
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