AssayMaxTM Human HSP47 ELISA Kit
Contents
1. 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 use supernatants and assay Store samples 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 Collect the serum and assay Store samples at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Lysates Place the cell culture dish in ice and wash the cells with ice cold PBS Drain the PBS then add ice cold lysis buffer 20 mM Tris HCl pH 7 5 150 mM NaCl 1 mM Na2EDTA 1 mM EGTA 1 Triton 0 1 mM PMSF 1 ug ml leupeptin 1 ug mL aprotinin and 1 ug mL pepstatin Scrape adherent cells off the dish and then transfer the cell suspension into a pre cooled microfuge tube Maintain constant agitation for 30 minutes at 4 C Centrifuge in a microcentrifuge at 4 C Collect fresh cell lysates Use undiluted sa2. 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 Biotinylated Human HSP47 Antibody to each well and incubate for 2 hours 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 25 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 tr
3. 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human HSP47 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 HSP47 Standard Human HSP47 in a buffered protein base 20 ng lyophilized 2 vials e Biotinylated Human HSP47 Antibody 100x A 100 fold concentrated biotinylated polyclonal antibody against HSP47 60 pl e EIA Diluent Concentrate 10x A 10 fold concentrated buffered protein base 20 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 concentrated 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 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
4. A assarbno AssayMax Human HSP47 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 2 hours 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 BE Consult instructions for use Assay Template 12 11 10 Human Heat Shock Protein 47 HSP47 ELISA Kit Catalog No EH5202 1 Sample insert for reference use only Introduction Heat shock protein of 47 kDa HSP47 also called serpin H1 clade H member 1 collagen binding protein 1 and collagen is a member of the serpin superfamily of serine proteinase inhibitors HSP47 is a 418 amino acids collagen specific molecular chaperone involved in the collagen folding and secretion It localizes to the endoplasmic reticulum lumen and its expression is induced by heat shock 1 2 Principle of the Assay The Ass
5. ayMax Human Heat Shock Protein 47 HSP47 ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human HSP47 in plasma serum milk tissue extract and cell culture lysates This assay employs a quantitative sandwich enzyme immunoassay technique that measures human HSP47 in less than 5 hours A polyclonal antibody specific for human HSP47 has been pre coated onto a 96 well microplate with removable strips HSP47 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for HSP47 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 used beyond the expiration date Reagents e Human HSP47 Microplate A
6. e plasma samples in one assay Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 3 25 ng ml Recovery 84 11196 Average Recovery 96 9696 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum No Dilution 9596 9396 1 2 10596 10296 1 4 11196 11196 Cross Reactivity Species Cross Reactivity Beagle None Bovine None Monkey lt 10 Mouse lt 10 Rat None Swine lt 50 Rabbit None References 1 Strausberg RL et al 2002 Proc Natl Acad Sci U S A 99 16899 16903 2 Razzaque MS et al 2005 Contrib Nephrol 148 57 69 Version 1 6R2 Related Products EH5001 1 AssayMax Human HSP27 ELISA Kit Plasma Serum Milk Tissue Extract and Cell Culture samples EH5505 1 AssayMax Human HSP60 ELISA Kit Plasma Serum and Cell Culture samples www assaypro com e e mail Support assaypro com
7. iplicate 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 Typical Data e The typical data is provided for reference only Individual laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point P1 50 00 Average OD P2 25 00 P3 12 50 P4 6 250 P5 3 125 P6 1 563 P7 0 000 Sample Sodium Citrate Plasma 1x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed OD 450 nm 0 1 Human HSP47 Standard Curve 10 100 H HSP47 ng ml Reference Value e Human plasma and serum samples from adults under mild amounts of daily stress and otherwise healthy were tested n 20 On average HSP47 level was 3 1 ng ml Performance Characteristics e The minimum detectable dose of HSP47 as calculated by 25D from the mean of a zero standard was established to be 0 9 ng ml e Intra assay precision was determined by testing replicates of thre
8. mples or dilute samples 1 2 with EIA Diluent and assay The undiluted samples can be stored at 20 C or below e Tissue Extract tissue samples with 50 mM phosphate buffered saline pH7 4 containing 1 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant and measure the protein concentration Use undiluted samples or dilute samples 1 2 with EIA Diluent and assay The undiluted samples can be stored at 20 C or below e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes and assay Store samples 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 20 ng of Human HSP47 Standard with 0 4 ml of EIA Diluent to generate a 50 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 50 ng ml 1 2 with EIA Diluent to produce 25 12 5 6 25 3 125 and 1 563 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solu
9. tion should be frozen at 20 C and used within 48 hours Standard Point Dilution HSP47 ng ml P1 1 part Standard 50 ng ml 50 00 1 part P1 1 part EIA Diluent 25 00 1 part P2 1 part EIA Diluent 12 50 P4 1partP3 1 part EIA Diluent 6250 Pe 1partPS 1partElADiluenn 156 e Biotinylated Human HSP47 Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 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 HSP47 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
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