AssayMaxTM Human ADAMTS13 Autoantibody ELISA Kit
Contents
1. Elevated Level Sample 8x Serum with elevated level of anti ADAMTS13 IgG Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Adamts 13 Autoantibody Standard Curve OD 450 nm 1 10 00 hAdamts13 IgG AU ml Reference Value e _ Human plasma and serum samples from healthy adults were tested n 20 Moreover patient serum samples containing high levels of anti ADAMTS13 IgG were tested n 9 The following ranges have been established Sample _Anti ADAMTS13 IgG AU ml Normal Level lt 10 0 Elevated Level gt 10 0 It is recommended that each laboratory establishes its own normal and pathological ranges of antibodies Performance Characteristics e The minimum detectable dose of autoantibodies as calculated by 2SD from the mean of a zero standard was established to be 0 3 AU ml e Intra assay precision was determined by testing replicates of three plasma samples in one assay e Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision 1 2 3 1 2 3 20 20 CV 20 20 20 20 Average CV Linearity e Serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Serum 1 2 90 1 4 Troubleshootin2. performed at room temperature 20 25 C 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 Add 50 ul of 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 HRP Conjugate to each well and incubate for 1 hour Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 20 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 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 ge
3. MA assarbro AssayMax Human ADAMTS13 Autoantibody ELISA Kit Anti ADAMTS13 IgG 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 HRP Conjugate per well Incubate 1 hour Step 3 Wash then add 50 ul of Chromogen Substrate per well Incubate 20 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 ADAMTS13 Autoantibody ELISA Kit Anti ADAMTS13 IgG Catalog No EA7550 1 Sample protocol for reference use only Introduction ADAMTS13 a disintegrin like and metalloproteinase with a thrombospondin type 1 motif 13 also called vonWillebrand factor cleaving protease VWFCP is the 13th member of the ADAMTS family of metalloproteases It is a multidomain protease synthesized in the liver and secreted into the blood where it cleaves von Willebrand factor vWF and thereby limits platelet thrombosis 1 2 ADAMTS13 encodes a mature 1 353 amino acid protein with a calculated 145 kDa and a gly
4. cosylated 190 kDa molecular mass In von Willebrand disease increased exposure of vWF to ADAMTS13 would predispose to bleeding by causing increased degradation of vWF Autoimmune inhibitory antibodies or genetic mutations cause deficiency of ADAMTS13 which leads to thrombotic thrombocytopenic purpura and acute and chronic inflammation 3 5 Autoantibodies against ADAMTS13 belong to the pANCA class Perinuclear Anti Neutrophil Cytoplasmic Antibodies Anti ADAMTS antibodies are found in patients with recurrent thrombotic thrombocytopenic purpura TTP 6 Principle of the Assay The AssayMax Human ADAMTS13 Autoantibody ELISA Enzyme Linked Immunosorbent Assay kit is designed for the quantitative determination of autoimmune response IgG to a target antigen ADAMTS13 The kit detects autoantibodies in plasma and serum samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures autoantibodies anti ADAMTS13 IgG in less than 4 hours An ADAMTS13 antigen has been pre coated onto a 96 well microplate with removable strips Autoantibody specific for ADAMTS13 in standards and samples is sandwiched by the immobilized antigen and an antibody HRP 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 Prepare all reagents working diluent buffer wash buffer standard HRP conjugat
5. e 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 HRP conjugate vial before opening and using contents e The Stop Solution is an acidic solution e This kit is for research use only e The kit should not be used beyond the expiration date Reagents e Human ADAMTS13 Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with ADAMTS13 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 ADAMTS13 Standard Plasma standard 50 AU lyophilized e HRP Conjugate 50x A 50 fold concentrated HRP antibody conjugate 120 yl 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 1 bottle 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 HRP Conjugate at 20 C e Store Microplate Diluent Concentrate 10x Wash Buffer Stop Soluti
6. g 97 Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components from different lots Improper wash step e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner Inconsistent volumes loaded into wells Low Precision e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Improperly sealed microplate e Check the microplate pouch for proper sealing e Check that the microplate pouch has no punctures e Check that three desiccants are inside the microplate pouch prior to sealing Microplate was left unattended between steps e Each step of the procedure should be performed uninterrupted Omission of step e Consult the provided procedure for complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calib
7. ion e Thoroughly mix dilutions References 1 Banno F et al 2009 Blood 113 21 5323 5329 2 Soejima Ket al 2001 J Biochem 130 4 475 480 3 Levy GG et al 2001 Nature 413 488 494 4 Furlan M et al 1998 N Engl J Med 339 22 1578 1584 5 Chauhan AK et al 2008 J Exp Med 205 9 2065 2074 6 Rahel Froehlich Zahnd et al 2012 Haematologica 97 2 297 303 doi 10 3324 haematol 2011 051433 PMCID PMC3269492 Version 1 0 Related Products e EL2550 1 AssayMax ADAMTS13 ELISA Kit Plasma Serum Saliva CSF and Cell Culture samples www assaypro com e mail Support assaypro com April 2015
8. ix 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 Standard 50 AU with 1 ml of EIA Diluent to generate a 50 AU ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate points by serially diluting the standard stock solution 50 AU ml 1 2 using equal volume of EIA Diluent to produce 25 12 5 6 25 3 125 and 1 563 AU ml solutions EIA Diluent serves as the zero standard 0 AU ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution ADAMTS13 AU ml P1 1 part Standard 50 AU ml 50 00 1 part P1 1 part EIA Diluent 25 00 1 part P2 1 part EIA Diluent 12 50 Pa 1partP3 1 part EIA Diluent 6250 Ps ipartPS 1partElADiluent 1563 HRP Conjugate 50x Spin down the HRP Conjugate briefly and dilute the desired amount of the conjugate 1 50 with ElA Diluent Any remaining solution should be frozen at 20 C 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 Assay Procedure Prepare all reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is
9. nerated 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 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 e Although normal samples have been diluted 1 4 do not multiply the value by the dilution factor Samples with elevated level of autoantibodies can be diluted further for example 1 8 Account for this further dilution factor when calculating the value of the sample Dilution Multiplication Factor For Factor Calculating Values Example Serum with normal level of anti ADAMTS13 IgG Serum with elevated level of anti ADAMTS13 IgG Serum with elevated level of anti ADAMTS13 IgG 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 Average OD P1 50 00 P2 25 00 P3 12 50 P4 6 250 P5 3 125 P6 1 563 P7 0 000 Normal Level Sample 4x Serum with normal level of anti ADAMTS13 IgG
10. on 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 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 4 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 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 4 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 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 m
11. ration e Check pipette for proper performance Wash step was skipped Unexpectedly Low or High Signal Intensity e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the assay in the incubator or at room temperature Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitut
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