Human Factor XII ELISA Kit
Contents
1. 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 1000 fold dilution Assuming the needed volume is less than or equal to 240 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than or equal to 240 ul Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e _ MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the MIX Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Human FXII Standard Reconstitute the 250 ng of Human FXII Standard with 2 5 ml of MIX Diluent to generate a 100 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 100 ng ml 1 4 with MIX Diluent to produce 25 6 25 1 563 0 391 0 098 and 0 024 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution FXII ng ml P1 1 part Standard 100 ng ml 100 0 1 part P1 3 parts MIX Diluent 25 00 1 part P2 3 parts MIX Diluent 6 250 p4 A part P3 3 parts MIX Diluent 1 563 Pe2. tpartPS 3parsMIXDiluent 0098 Ps mixpiun 0000 e Biotinylated Human FXII Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with MIX 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 MIX 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 XII 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 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 th
3. 4 assaPro AssayMax Human Factor XII ELISA Kit Assaypro LLC 3400 Harry S Truman Blvd St Charles MO 63301 T 636 447 9175 F 636 447 9475 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 1 hour 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 30 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 Factor XII ELISA Kit Catalog No EF1012 1 Sample insert for reference use only Introduction Human coagulation factor XII FXII Hageman factor is a plasma serine protease existing in the zymogen form Upon contact with negatively charged artificial or biologic surfaces FXII is autoactivated into FXlla that initiates intrinsic blood coagulation fibrinolysis and activation of the inflammatory kallikrein kinin and complement systems 1 3 FXII has 615 amino acids weighs 80 kDa and circulates in normal plasma at a concentration of 30 ug m
4. ctor 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 e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Schousboe 1990 Eur J Biochem 193 495 499 2 Schmaier AH 2008 J Clin Invest 118 3006 3009 3 Maas C etal 2008 J Clin Invest 118 3208 3218 4 Reddigari SR et al 1993 J Biol Chem 268 11982 11987 5 Kaplan AP and Silverberg M 1987 Blood 70 1 15 6 Mahdi F et al 2002 Blood 99 3585 3596 Version 2 2R www assaypro com e e mail Support assaypro com
5. e 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 0 098 25 ng ml Recovery 91 111 Average Recovery 99 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 500 98 98 1 1000 99 100 1 2000 103 104 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey 5 Mouse None Rat None Swine None Rabbit None Human 100 Protein Cross Reactivity Human Factor XIla 100 Troubleshooting 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 e Check that the correct wash buffer is being used S e Check that all wells are dry after aspiration a Improper wash step e Check that the microplate washer is dispensing properly D e If washing by pipette check for proper pipetting a technique 2 Splashing of reagents e Pipette properly in a controlled and careful manner 2 while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Inconsistent volumes loaded into wells e Thoroughly agitate the lyophilized components after reconstitution
6. e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Insufficient mixing of reagent dilutions Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Microplate was left e Each step of the procedure should be performed T unattended between uninterrupted En steps a Omission of step e Consult the provided procedure for complete list of steps Steps performed in e Consult the provided procedure for the correct order incorrect order 5 z Insufficient amount of e Check pipette calibration 2 3 reagents added to e Check pipette for proper performance a wells 2s Wash step was skipped e Consult the provided procedure for all wash steps 3 Improper wash buffer e Check that the correct wash buffer is being used E Improper reagent e Consult reagent preparation section for the correct preparation dilutions of all reagents z Insufficient or e Consult the provided procedure for correct incubation gt prolonged incubation time periods e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution fa
7. e plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Human FXII 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 30 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 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 anal
8. l 4 5 It is a multidomain protein with structure similarity to EGF single chain urokinase and tissue plasminogen activator In the intravascular compartment FXII binds to endothelial cell urokinase plasminogen activator receptor cytokeratin 1 and the complement receptor 6 Principle of the Assay The AssayMax Human Factor XII FXII ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human factor XII in plasma serum milk urine CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures FXII in 4 hours A murine antibody specific for FXII has been pre coated onto a 96 well microplate with removable strips FXII in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for FXII 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 determi
9. nd 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 and collect supernatants Dilute samples 1 1000 with MIX Diluent or within the range of 1 500 to 1 5000 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 1000 with MIX Diluent or within the range of 1 500 to 1 5000 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 f
10. ne 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 FXII Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a murine antibody against FXII 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 FXII Standard Human FXII in a buffered protein base 250 ng lyophilized e Biotinylated Human FXII Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against FXII 140 pul e _ MIX 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 pl 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 a
11. or 10 minutes at 4 C to remove debris Collect supernatants and assay The samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Urine Collect urine 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 e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Milk dilution is recommended for use at 1 4 in MIX Diluent however depending on application needs user should determine proper dilutions 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 10 into MIX 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 1 100 1 10000 A 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is less than or equal to 400 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than or equal to 400 ul 1 1000 1 100000
12. ysis 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 Average OD P1 100 0 P2 25 00 P3 6 250 P4 1 563 PS 0 391 P6 0 098 P7 0 024 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 1000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed fXII Standard Curve 1 07 OD 450 nm 0 1 T T T 0 1 1 0 10 0 100 0 FXI ng ml Reference Value e Human plasma and serum samples from healthy adults were tested n 30 On average factor XII level was 29 ug ml Sample Average Value ug ml Human Pool Normal Plasma 25 9 Human Pool Normal Serum 32 5 Performance Characteristics e The minimum detectable dose of factor XII as calculated by 2SD from the mean of a zero standard was established to be 0 02 ng 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 Sampl
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