AssayMaxTM Rat Ceruloplasmin ELISA Kit
Contents
1. 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 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 25 ug of Rat Ceruloplasmin Standard with 0 5 ml of MIX Diluent to generate a 50 ug ml standard 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 solution 50 ug ml 1 2 with MIX Diluent to produce 25 12 5 6 25 3 125 1 563 and 0 781 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 Rat Ceruloplasmin Point ug ml Standard 50 ug ml 50 00 1 part P1 1 part MIX Diluent 25 00 1 part P2 1 part MIX Diluent 12 50 Pa 1partP3 1partMixDiluent 6250 Ps tart PS ipartMixDiluent 1563 P8 MikxDilent 000 e Biotinylated Rat Ceruloplasmin 1x Reconstitute Biotinyla2. AssayMax Human Ceruloplasmin ELISA Kit Plasma and Serum Samples ERC4101 1 AssayMax Rat Ceruloplasmin ELISA Kit Urine and Cell Culture samples www assaypro com E mail Support assaypro com
3. the Assay The AssayMax Rat Ceruloplasmin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of rat ceruloplasmin in plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures rat ceruloplasmin in less than 3 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 competed by a biotinylated ceruloplasmin 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 RatCeruloplasmin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclo
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 25 ul of Standard or Sample and 25 ul of Biotinylated Protein per well Incubate 2 hours 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 15 minutes Step 4 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key BE Consult instructions for use Assay Template 12 11 10 Rat Ceruloplasmin ELISA Kit Catalog No ERC4001 1 Sample insert for reference use only Introduction Ceruloplasmin is an abundant alpha 2 serum glycoprotein that contains 95 ofthe 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 Ceruloplasmin concentration on average 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 region of the brain 1 3 5 Principle of
5. d 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 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 advance Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 15 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 Add 50 ul of Stop Solution to each well The color will change from blue to yellow 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 Calculate the mean value of the duplicate or triplicate readings for each standard and sample 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 curv
6. e fit Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Standard Curve The curve is provided for illustration only A standard curve should be generated each time the assay is performed Rat Ceruloplasmin Standard Curve 1 0 OD450 nm 0 1 ri Ceruloplasmin ug ml 1 ol 10 10 Precision Sensitivity and Specificity The minimum detectable dose of rat ceruloplasmin is typically 0 7 ug ml Intra assay and inter assay coefficients of variation were 4 4 and 7 0 respectively Linearity Sample Dilution Plasma Serum 1 200 89 90 1 400 99 98 1 800 10496 103 Recovery Standard Added Value 1 20 ug ml Recovery 83 111 Average Recovery 96 Cross Reactivity Species Cross Reactivity Beagle None Bovine None Monkey None Mouse lt 5 Human None Swine None References 1 2 3 4 5 Harris Z L etal 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 Jetal J Clin Invest Volume 80 October 1987 1200 1204 Kumar A et al WJM October 1995 Vol 163 No 4 Moller L B etal 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 EC4001 1
7. er 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 1 400 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 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 1 400 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
8. nal 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 25 ug lyophilized e Biotinylated Rat Ceruloplasmin 1 vial lyophilized 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 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 read
9. ted Rat Ceruloplasmin with 4 ml MIX Diluent to produce a working solution Allow the biotin to sit for 10 minutes with gentle agitation prior to use 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 Add25 ul of Rat Ceruloplasmin Standard or sample per well and immediately add 25 ul of Biotinylated Rat Ceruloplasmin 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 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 liqui
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