AssayMax Human Factor XI ELISA Kit
Contents
1. 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 Factor XI Antibody to each well and incubate for 1 hour e Wash the microplate as described above e 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 e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 15 minutes or till the optimal color density develops Gently tap the 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 for each standard and sample e To generate a stand2. mU ml Standard 50 ng ml 50 00 6 200 1 part P1 1 part EIA Diluent 25 00 3 100 1 part P2 1 part ElA Diluent 12 50 1 550 P4 1partP3 1 part EIA Diluent 6250 0775 Pe 1partP5 1 part EIA Diluent 1562 0 194 e Biotinylated Human Factor XI 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 Factor XI 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
3. through feedback activation linking to tissue factor or extrinsic pathway FXla in turn cleaves factor IX and triggers a cascade event converting fibrinogen to a stable cross linked fibrin clot formation 1 3 FXI also plays a role in the prevention of clot lysis from fibrinolysis 4 Congenital FXI deficiency is accompanied by mild and injury related bleeding Severe FXI deficiency is linked to low occurrence of ischemic stroke or venous thrombosis 5 In contrast elevated FXI activity is a risk factor for stroke venous thrombosis and coronary artery disease 6 8 FXI is a new target for the treatment and prevention of thromboembolism Principle of the Assay The AssayMax Human Factor XI FXI ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human factor XI in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures FXI in less than 4 hours A polyclonal antibody specific for FXI has been pre coated onto a 96 well microplate with removable strips FXI in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for FXI 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 d
4. A assarPno Human Factor XI 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 2assaypro 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 I 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 15 minutes Il Add 50 ul of Stop Solution per well Read at 450 nm immediately Assay Template 12 11 10 AssayMax Human Factor XI ELISA Kit Catalog No EF1011 1 Sample protocol for reference only Introduction Human coagulation factor XI FXI also called plasma thromboplastin antecedent is a serine protease important for initiating the contact activation or intrinsic pathway of blood coagulation FXI is present in plasma as a homodimer zymogen consisting of two identical polypeptide chains of 607 amino acids and 80 kDa each FXI circulates in normal plasma at a concentration of 5 ug ml It is activated to form FXla not only by factor XIla through the contact pathway but also by thrombin
5. ard 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 Human FXI Standard Curve OD 450 nm 0 1 10 10 0 f XI mU ml Human FXI Standard Curve OD 450 nm 1 0 10 0 100 0 f XI ng ml Performance Characteristics e The minimum detectable level of FXI was typically 1 5 ng ml Intra assay and inter assay coefficients of variation were 5 1 and 7 1 respectively e Kit standard has been calibrated against WHO International Standard Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 300 90 92 1 600 99 99 1 1200 104 103 Recovery Standard Added Value 3 13 25 ng ml Recovery 84 112 Average Recovery 97 5 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey None Mouse None Rat None Swine None Rabbit None Proteins Cross Reactivity Factor XI 10096 Factor Xla 10096 Reference Value Normal plasma FXI levels rang
6. e from 0 4 IU ml 1 6 IU ml 3200 ng ml 12000 ng ml References 1 2 3 4 5 6 7 8 Fujikawa K et al 1986 Biochemistry 25 2417 2424 Asakai R et al 1987 Biochemistry 26 7221 7228 Samuel D et al 2007 Proc Natl Acad Sci U S A 104 15693 15698 von dem Borne PA et al 1995 Blood 86 3035 3042 Salomon O et al 2008 Blood 111 4113 4117 Yang DT et al 2006 Am J Clin Pathol 126 411 415 Eichinger S et al 2004 Blood 103 3773 3776 Berliner Jl et al 2002 Thromb Res 107 55 60 Version 3 0 www assaypro com e E mail Support assaypro com
7. iluent 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 vial before opening and using contents e This kit is for research use only e Thekit should not be used beyond the expiration date e The Stop Solution is an acidic solution Reagents e Human Factor XI Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against FXI 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 Factor XI Standard Human FXI in a buffered protein base 50 ng lyophilized 2 vials e Biotinylated Human Factor XI Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against FXI 140 ul 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 concentrated 80 ul e Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution A 0 5 N h
8. mples at 3000 x g for 10 minutes and remove serum Dilute samples 1 600 into EIA Diluent or within the range of 1 300 to 1 1200 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 Collect cell culture media and centrifuge at 3000 x g for 10 minutes at 4 C to remove debris Collect supernatants and assay 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 Human Factor XI Standard Reconstitute the 50 ng 6 2 mU of Human Factor XI Standard with 1 ml of EIA Diluent to generate a 50 ng ml 6 2 mU 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 50 ng ml 1 2 with EIA Diluent to produce standard solution of 25 12 5 6 25 3 125 and 1 562 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 5 days Standard FXI FXI Point ng ml
9. ydrochloric 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 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 and collect supernatants Dilute samples 1 600 into EIA Diluent or within the range of 1 300 to 1 1200 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 sa
Download Pdf Manuals
Related Search