Human Neutrophil Elastase ELISA Kit
Contents
1. 00 1 10000 A 4ulsample 396 ul buffer A 4ulsample 396 ul buffer 100x 100 fold dilution B 4ulofA 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 pul 1 1000 1 100000 A 4ulsample 396 ul buffer 100x A 4ulsample 396 ul buffer 100x B 24yglofA 216 ul buffer 10x B 4ulofA 396 ul buffer 100x 1000 fold dilution C 24JylofB 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 pul 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 20 ng of Human Neutrophil Elastase Standard with 2 ml of EIA Diluent to generate a 10 ng 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 10 ng ml 1 2 with EIA Diluent to produce 5 2 5 1 25 0 625 0 313 and 0 156 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solut2. A assarbno AssayMax Human Elastase 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 U 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 7 minutes L Add 50 ul of Stop Solution per well Read at 450 nm immediately Assay Template 12 11 10 Human Neutrophil Elastase ELISA Kit Catalog No EE1001 1 Sample insert for reference use only Introduction Human Neutrophil Elastase NE also known as ELA2 is a subfamily of serine proteinase elastases It consists of 218 amino acid residues with a molecular weight of 23 kDa and two asparagine linked carbohydrate side chains 1 Neutrophil elastase is stored in neutrophil lysosomes azurophil granules during neutrophil differentiation and is involved in a variety of immune defense reactions and degenerative and inflammatory disorders 2 3 Upon infection the activated neut
3. ad Sci U S A 89 17 8215 8219 4 Takahashi H et al 1988 J Biol Chem 263 29 14739 14747 5 Belaaouaj A et al 2000 Science 289 5482 1185 1188 6 Weinrauch Y et al 2002 Nature 417 6884 91 94 Version 2 5R www assaypro com e E mail Support assaypro com
4. ant 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 Neutrophil Elastase Antibody to each well and incubate for 1 hour e Wash the microplate as described above 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 7 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 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 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 s
5. ion should be frozen at 20 C and used within 30 days StandardPoint Dilution Elastase ng ml pa ipatP3ripattADiuen 1250 e ipatPSripattADiuem 0313 tiene 900 e Biotinylated Neutrophil Elastase Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with EIA 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 EIA 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 Human Neutrophil Elastase 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 dec
6. n 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 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 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 50 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 50 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 e Cell Culture Supernatants Centrifuge cell cul
7. rophils release elastase which then hydrolyzes azurophil granule proteins and extracellular matrix proteins elastin collagen and proteoglycan 4 Asa host defense protein neutrophil elastase degrades bacterial outer membranes and virulence proteins 5 6 Principle of the Assay The AssayMax Human Neutrophil Elastase ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human neutrophil elastase in plasma serum urine saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human elastase in less than 4 hours A polyclonal antibody specific for human elastase has been pre coated onto a 96 well microplate with removable strips Neutrophil elastase in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for elastase 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 sugge
8. sted 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 kit should not be used beyond the expiration date e The Stop Solution is an acidic solution Reagents e Human Neutrophil Elastase Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human neutrophil elastase 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 Neutrophil Elastase Standard Human neutrophil elastase in a buffered protein base 20 ng lyophilized e Biotinylated Neutrophil Elastase Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against neutrophil elastase 140 pul 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 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 expiratio
9. tandard 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 four parameter or log log 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 Human Elastase Standard Curve o T OD 450 nm 0 1 1 1 1 107 10 10 hElastase ng ml Sensitivity and Specificity e The minimum detectable dose of elastase is typically 0 15 ng ml e Intra assay and inter assay coefficients of variation were 5 0 and 7 1 respectively Linearity Sample Dilution Plasma Serum 1 25 106 94 1 50 101 97 1 100 99 108 Recovery Standard Added Value 0 5 5 ng ml Recovery 96 84 113 Average Recovery 96 9696 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey 1096 Mouse None Rat None Swine None Rabbit None Human 10096 Reference Value e Normal human elastase plasma levels are 160 ng ml References 1 SinhaS et al 1987 Proc Natl Acad Sci U S A 84 8 2228 2232 2 Lindmark A et al 1990 Blood 76 11 2374 2380 3 Zimmer M et al 1992 Proc Natl Ac
10. ture 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 urine 1 8 with 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 e Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute saliva 1 1000 with 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 e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute milk 1 200000 with EIA Diluent or within the range 1 100000 to 1 400000 and assay The user should determine the optimal dilution factor 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 CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 20 into ElA 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 1
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