AssayMax Mouse Hemopexin ELISA Kit
Contents
1. 2004 Nippon Rinsho 62 3 577 86 Version 1 1 Related Products EH1001 1 AssayMax Human Hemopexin ELISA Kit Plasma and Serum samples EH2001 1 AssayMax Human Hemopexin ELISA Kit Urine Milk Saliva CSF and Cell Culture samples ERH2001 1 AssayMax Rat Hemopexin ELISA Kit Plasma Serum Urine and Cell Culture samples www assaypro com e E mail Support assaypro com2. Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 5 into MIX Diluent or within the range of 2x 20x and assay The undiluted samples can be stored at 20 C or below for up to 3 months 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 Mouse Hemopexin Standard Reconstitute the 24 ng of Mouse Hemopexin Standard with 1 ml of MIX Diluent to generate a 24 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 24 ng ml 1 2 with MIX Diluent to produce 12 6 3 1 5 0 75 0 375 and 0 188 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and use within 30 days Standard Mouse Hemopexin Point ng ml 1 part Standard 24 ng ml 1 part MIX Diluent 12 09 1 part P1 1 part MIX Diluent 6 000 1 part P2 1 part MIX Diluent 3 000 Pa 1partP3 1 part MIX Diluent 1500 Ps 1partP5 1 part MIX Dilu
3. 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 T OD 450 nm Mouse Hemopexin Standard Curve 0 1 0 1 Performance Ch 1 0 10 0 100 0 Mouse Hemopexin ng ml aracteristics e The minimum detectable dose of mouse hemopexin is typically 0 1 ng ml e Intra assay and inter assay coefficients of variation were 4 9 and 7 2 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 100000 88 90 1 200000 96 97 1 400000 106 104 Average Percentage of Expected Value Sample Dilution Urine 1 2 85 1 5 95 1 20 103 Recovery Standard Added Value 0 3 3 0 ng ml Recovery 84 114 Average Recovery 96 96 596 Cross Reactivity Species Cross Reactivity Canine None Monkey None Mouse 10096 Rat 5096 Rabbit None Swine None Human None References 1 2 3 4 5 Delanghe JR et al 2001 Clin Chim Acta 312 1 2 13 23 Bakker WW et al 2005 Pediatr Nephrol 20 10 1410 5 Yu HL et al 2003 Proteomics 3 11 2240 8 Kristensson Aas A et al 1986 Eur J Clin Invest 16 2 178 83 Suzuki K et al
4. Wyssaypro Mouse Hemopexin 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 Symbol Key Consult instructions for use Assay Summary Add 50 ul of Standard Sample per well Incubate 2 hours 1 Wash then add 50 ul of Biotinylated Antibody per well Incubate 1 hour Jl Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes U Wash then add 50 ul of Chromogen Substrate per well Incubate 10 minutes 1 Add 50 ul of Stop Solution per well Read at 450 nm immediately Assay Template 12 11 10 AssayMax Mouse Hemopexin ELISA Kit Catalog No EMH2001 1 Sample protocol for reference only Introduction Hemopexin is a heme binding plasma glycoprotein which after haptoglobin forms the second line of defense against hemoglobin mediated oxidative damage during intravascular hemolysis A decrease in plasma hemopexin concentration reflects a recent release of heme compounds in the extracellular compartment Heme hemopexin complexes are delivered to hepatocytes by receptor mediated endocytosis after which hemopexin is recycled to the circulation 1 Studies indicate that increased hemope
5. ate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 daysina vacuum desiccator Diluent 1x may be stored for up to 30 days at 2 8 C Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required 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 and Storage 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 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 200000 into MIX Diluent or within the range of 50000x 500000x 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 200000 into MIX Diluent or within the range of 50000x 500000x and assay The undiluted samples can be stored at 20 C or below for up to 3 months
6. ent 0375 P8 MXDilent 0000 e Biotinylated Mouse Hemopexin 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 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 Mouse Hemopexin 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 decanti
7. ial before opening and using contents This kit is for research use only The kit should not be used beyond the expiration date The Stop Solution is an acidic solution Reagents Mouse Hemopexin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against mouse hemopexin Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes which can be cut to fit the format of the individual assay Mouse Hemopexin Standard Mouse hemopexin in a buffered protein base 24 ng lyophilized Biotinylated Mouse Hemopexin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against mouse hemopexin 140 ul MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store SP Conjugate and Biotinylated Antibody at 20 C Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C Unused micropl
8. ng the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Mouse Hemopexin Antibody to each well and incubate for 1 hour e 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 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 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 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 on the y axis The best fit line can be determined by regression analysis using log
9. xin levels are associated with minimal change disease MCD 2 sporadic Alzheimer s disease AD 3 heavy drinking chronic alcoholics 4 hemolytic anemias chronic neuromuscular diseases and acute intermittent porphyria 5 Principle of the Assay The AssayMax Mouse Hemopexin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of mouse hemopexin in plasma serum urine and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse hemopexin in less than 4 hours A polyclonal antibody specific for mouse hemopexin has been pre coated onto a 96 well microplate with removable strips Mouse hemopexin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for mouse hemopexin 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 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 protocol However the user should determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody v
Download Pdf Manuals
Related Search