AssayMaxTM Mouse Fibrinogen ELISA Kit
Contents
1. 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 25 ul of Mouse FBG Standard or sample per well and immediately add 25 ul of Biotinylated Mouse FBG to each well on top of the standard or sample and tap plate to mix gently 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 the plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Streptavidin Peroxidase Conjugate to each well and in2. receptor to form bridges between platelets thus facilitating aggregation 2 Principle of the Assay The AssayMax Mouse Fibrinogen ELISA Enzyme Linked Immunsorbent Assay kit is designed for detection of mouse FBG in plasma samples This assay employs a quantitative competitive enzyme immunoassay technique that measures mouse FBG in less than 3 hours A polyclonal antibody specific for mouse FBG has been pre coated onto a 96 well microplate with removable strips FBG in standards and samples is competed with a biotinylated mouse FBG sandwiched by the immobilized antibody and 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 protein 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 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 Mouse FBG Microplate A 96 well polystyrene microplate 12 str
3. agents 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 Standard Curve Reconstitute the 100 ug of Mouse FBG Standard with 2 5 ml of MIX Diluent to generate a 40 ug 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 40 ug ml 1 2 with equal volume of MIX Diluent to produce 20 10 5 2 5 and 1 25 ug ml solutions MIX Diluent serves as the zero standard 0 ug ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution Mouse FBG ug ml P1 1 part Standard 40 ug ml 40 00 1 part P1 1 part MIX Diluent 20 00 1 part P2 1 part MIX Diluent 10 00 Pa 1partP3 1 part MIX Diluent 5000 PG 1partP5 1 part MIX Diluent 1250 e Biotinylated Mouse FBG 4x Reconstitute Biotinylated Mouse FBG with 4 ml MIX Diluent to produce a 4 fold stock solution Allow the biotin to sit for 10 minutes with gentle agitation prior to making dilutions The stock solution should be further diluted 1 4 with MIX Diluent Any remaining solution should be frozen at 20 C and used within 30 days e Wash Buffer Concentrate 20x If
4. cubate 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 20 minutes or until the optimal 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 low 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 standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm absorbance OD 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
5. e Rat None Swine None Rabbit None Mouse 100 References 1 Doolittle R F 1984 Annu Rev Biochem 53 195 2 Handley D A and Hughes T E 1997 Thromb Res 87 1 Version 4 3R Related Products e _ EF1040 1 AssayMax Human Fibrinogen ELISA Kit Plasma samples EF2040 1 AssayMax Human Fibrinogen ELISA Kit Urine Milk Saliva and Cell Culture samples e ERF1040 1 AssayMax Rat Fibrinogen ELISA Kit Plasma samples e ERF2040 1 AssayMax Rat Fibrinogen ELISA Kit Urine and Cell Culture samples e EMF2040 1 AssayMax Mouse Fibrinogen ELISA Kit Urine and Cell Culture samples e _ ECF1040 1 AssayMax Canine Fibrinogen ELISA Kit Plasma samples e _ ECF2040 1 AssayMax Canine Fibrinogen ELISA Kit Urine and Cell Culture samples www assaypro com e e mail Support assaypro com
6. ips of 8 wells coated with a polyclonal antibody against mouse FBG Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Mouse FBG Standard Mouse FBG in a buffered protein base 100 ug lyophilized Biotinylated Mouse FBG 1 vial lyophilized MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 1 bottle Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store SP Conjugate at 20 C Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C 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 Diluent 1x may be stored for up to 30 days at 2 8 C Store Standard and Biotinylated Protein at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required Microplate reader capable of mea
7. suring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel 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 use supernatants Dilute samples 1 1000 into MIX 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 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 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 100 fold dilution 4 ul of A 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 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 4 ul of A 396 ul buffer 100x 1000 fold dilution 24 ul of B 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 ul Reagent Preparation e Freshly dilute all reagents and bring all re
8. values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 40 00 P2 20 00 P3 10 00 P4 5 000 P5 2 500 P6 1 250 P7 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 Mouse FBG Standard Curve E 1 0 o va st A O 0 1 1 pit 1 ait 1 ait L 10 10 10 10 mFBG ug ml Performance Characteristics e The minimum detectable dose of mouse fibrinogen as calculated by 25D from the mean of a zero standard was established to be 1 ug 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 Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 2 5 20 ug ml Recovery 85 109 Average Recovery 98 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma 1 500 106 1 1000 99 1 2000 94 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey None Human Non
9. yssaypro AssayMax Mouse Fibrinogen 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 25 ul of Standard or Sample and 25 ul of Biotinylated Protein per well Incubate 2 hours Step 2 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes 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 ci Consult instructions for use Assay Template 12 11 10 Mouse Fibrinogen FBG ELISA Kit Catalog No EMF1040 1 Sample insert for reference use only Introduction Fibrinogen FBG is a homodimer 340 kDa that is made up of two sets of alpha beta and gamma polypeptide chains FBG is synthesized in the parenchymal cell of the hepatocyte and in the megakaryocyte 1 FBG plays a major role in coagulation Upon cleavage by thrombin in the initial stages of coagulation activation FBG self assembles to yield a fibrin clot matrix that subsequently is crosslinked by factor Xllla to form an insoluble network FBG also binds to the platelet glycoprotein IIbllla
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