AssayMax Human Ceruloplasmin ELISA Kit
Contents
1. Standard with 3 ml of MIX Diluent to generate a 320 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 320 ng ml 1 4 with MIX Diluent to produce 80 20 5 and 1 25 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 14 days Standard ore Ceruloplasmin Dilution Point ng ml P1 1 part Standard 320 ng ml 320 0 1 part P1 3 parts MIX Diluent 80 00 P4 1partP3 3 parts MIX Diluent 5000 Pre MiXDient 000 e Biotinylated Human Ceruloplasmin 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 e2. 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 Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 10 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 Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 16 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 Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 1000 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 CSF Collect cerebrospinal fluid
3. CSF using sample pot 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 80 C 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 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 960 ng of Human Ceruloplasmin
4. 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 Ceruloplasmin Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition 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 Add 50 ul of Biotinylated Human Ceruloplasmin Antibody to each well and incubate for 1 hour Wash the microplate as described above 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 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 the 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 correc
5. anglia region of the brain 1 3 5 Principle of the Assay The AssayMax Human Ceruloplasmin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of ceruloplasmin in human urine saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures ceruloplasmin in less than 4 hours A polyclonal antibody specific for 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 a biotinylated polyclonal antibody specific for ceruloplasmin 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 e The kit should not be u
6. d assarbno AssayMax Human 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 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 Ceruloplasmin ELISA Kit Catalog No EC4101 1 Sample insert for reference use only Introduction Ceruloplasmin is an abundant alpha 2 serum glycoprotein that contains 95 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 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 g
7. sed beyond the expiration date Reagents e Human Ceruloplasmin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human 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 Human Ceruloplasmin Standard Human ceruloplasmin in a buffered protein base 960 ng lyophilized e Biotinylated Human Ceruloplasmin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human ceruloplasmin 140 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
8. tion 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 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 OD on the y axis The best fit line can be determined by regression analysis using log log or four parameter logistic curve 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 10 00 OD 1 00 450 nm 0 10 0 01 Ceruloplasmin Standard Curve ao iil go il po iii 1 10 100 1000 Ceruloplasmin ng ml Performance Characteristics e The minimum detectable dose of ceruloplasmin is typically 1 ng ml e Intra assay and inter assay coefficients of variation were 4 1 and 7 1 respectively Linearity Average Percentage of Expected Value Sample Dilution Milk 1 500 89 1 1000 96 1 2000 Recovery 99 Standard Added Val
9. ue 1 25 80 ng ml Recovery 85 116 Average Recovery 96 98 596 Cross Reactivity Species Cross Reactivity Beagle None Bovine None Monkey lt 70 Mouse None Rat None Swine lt 10 Rabbit None Human 100 References 1 2 3 4 5 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 Jet 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 2 0R Related Products EC4001 1 AssayMax Human Ceruloplasmin ELISA Kit Plasma and Serum samples ERC4101 1 AssayMax Rat Ceruloplasmin ELISA Kit Urine and Cell Culture samples ERC4001 1 AssayMax Rat Ceruloplasmin ELISA Kit Plasma and Serum samples www assaypro com e e mail Support assaypro com
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