AssayMaxTM Human Protein C ELISA Kit
Contents
1. 1 8 97 100 1 16 89 91 Recovery Standard Added Value 0 1 3 0 ug ml Recovery 81 108 Average Recovery 96 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 50 Mouse None Rat lt 15 Swine lt 50 Human 100 Rabbit None Reference Value e The normal blood level of protein C is 4 ug ml References 1 Foster DC et al 1985 Proc Natl Acad Sci U S A 82 14 4673 4677 2 Kisiel W etal 1976 Biochemistry 15 4893 4900 3 Esmon CT and Owen WG 1981 Proc Natl Acad Sci U S A 78 2249 2252 4 Wittletal 1994 Blood Coagul Fibrinolysis 5 4 651 653 5 Dahlback B et al 1993 Proc Natl Acad Sci U S A 90 1004 1008 6 Hansson PO etal 2000 Arch Intern Med 160 769 774 7 Mosnier LO et al 2007 Blood 109 8 3161 3172 Version 1 4R www assaypro com e E mail Support assaypro com2. 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 8 into EIA 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 8 into ElA Diluent 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 EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 4 5 ug of Human Protein C Standard with 0 75 ml of EIA Diluent to generate a 6 ug ml standard solution Allow the standard to
3. and performs a major role in regulating blood clotting inflammation and apoptosis 1 7 Principle of the Assay The AssayMax Human Protein C ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human protein C in plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures human protein C in less than 3 hours A polyclonal antibody specific for human protein C has been pre coated onto a 96 well microplate with removable strips Protein C in standards and samples is competed with a biotinylated protein C 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 Protein
4. C Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human protein C 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 Protein C Standard Human protein C standard in a buffered protein base 4 5 ug Iyophilized e Biotinylated Human Protein C 1 vial lyophilized e EIA 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 daysina 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
5. MA assarpro AssayMax Human Protein C 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 Human Protein C ELISA Kit Catalog No EP1311 1 Sample insert for reference use only Introduction Protein C is a vitamin K dependent plasma antithrombotic and anti inflammatory zymogenic glycoprotein that is synthesized in the liver Protein Chas a light chain of 155 amino acids 21 kDa and a heavy chain of 262 amino acids 41 kDa linked by a disulfide bond On endothelial cell membrane thrombin thrombomodulin complex cleaves a 12 residue peptide from protein C amino terminus of the heavy chain and converts it to activated protein C APC APC inactivates coagulation Factor Va and Factor Villa
6. d 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 Protein C Standard or sample per well and immediately add 25 ul of Biotinylated Human Protein C to each well on top of the standard or sample and 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 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 advance e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for about 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 e Add 50 ul of Stop Solution to each well The color will change from blue to y
7. ellow 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 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 H Protein C Standard Curve 1 0 ODaso nm 0 1F 1 l 107 10 107 hProtein C g ml Performance Characteristics e The minimum detectable dose of protein C is typically 0 09 ug ml e Intra assay and inter assay coefficients of variation were 4 8 and 7 3 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 4 95 105
8. sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting the standard solution 6 ug ml 1 2 with EIA Diluent to produce 3 1 5 0 75 0 375 0 188 and 0 094 ug ml solutions EIA Diluent serves as the zero standard 0 ug ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution Protein C ug ml P1 Standard 6 ug ml 6 000 1 part P1 1 part EIA Diluent 3 000 1 part P2 1 part ElA Diluent 1 500 P4 1partP3 ipartElADiluent 0750 Pe 1partPS ipartElADiluent 018 Ll PB ElADiluent 000 e Biotinylated Human Protein C 2x Reconstitute Biotinylated Human Protein C with 4 ml EIA Diluent to produce a 2 fold stock solution Allow to sit for 10 minutes with gentle agitation prior to making dilutions The stock solution should be further diluted 1 2 with EIA 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 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 EIA Diluent Any remaining solution should be frozen at 20 C Assay Procedure e Prepare all reagents standard solutions and samples as instructe
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