AssayMaxTM Human Fibronectin ELISA Kit
Contents
1. anticoagulant Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 100 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 can also be used as an anticoagulant Heparin is not recommended e Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 100 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 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 al2. 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 10 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 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 values listed Variations between laborato
3. ated Human FN 1x 1 vial lyophilized 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 e _ Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 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 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 daysina vacuum desiccator e Diluent 1x may be stored for up to 30 days at 2 8 C e _ Store Standard and Biotinylated Protein 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
4. ficient mixing of reconstitution reagent dilutions e Thoroughly mix dilutions References 1 2 3 4 5 Hynes R O 1992 Cell 69 11 Wu C etal 1995 Cell 83 715 Brown L F etal 1993 Am J Pathol 142 793 Pagani F etal 1991 J Cell Biol 113 1223 Verfaillie C M etal 1991 J Exp Med 174 693 Version 7 1R Related products EF2045 1 AssayMax Human Fibronectin ELISA Kit Urine Milk Saliva and Cell Culture samples EMF1045 1 AssayMax Mouse Fibronectin ELISA Kit Plasma Serum Urine and Cell Culture samples ERF1045 1 AssayMax Rat Fibronectin ELISA Kit Plasma Serum Urine and Cell Culture samples www assaypro com e mail Support assaypro com
5. ix 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 Human FN Standard or sample per well and immediately add 25 ul of Biotinylated Human FN 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 incubate for 30 minutes Turn on the microplate
6. l 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 Standard Curve Reconstitute the 25 ug of Human FN Standard with 0 5 ml of MIX Diluent to generate a 50 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 50 ug ml 1 2 with MIX Diluent to produce 25 12 5 6 25 3 125 1 563 and 0 781 ug ml solutions MIX Diluent serves as the zero standard 0 pg ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution FN ug ml P1 1 part Standard 50 ug ml 50 00 1 part P1 1 part MIX Diluent 25 00 1 part P2 1 part MIX Diluent 12 50 Pa 1partP3 1 part MIX Diluent 6250 Pe 1partP5 1 part MIX Diluent 1563 Ll PB mMiDilen 0000 e Biotinylated Human FN 1x Reconstitute Biotinylated Human FN with 4 ml MIX Diluent to produce a stock solution Allow the biotin to sit for 10 minutes with gentle agitation prior to use Any remaining solution should be frozen at 20 C and used within 30 days e Wash Buffer Concentrate 20x If crystals have formed in the concentrate m
7. orrect order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped 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 reagents e A new tip must be used for each addition of different samples or reagents during the assay procedure Contents of wells evaporate e Verify that the sealing film is firmly in place before placing 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 e Thoroughly agitate the lyophilized components after Insuf
8. oubleshooting Causes Course of Action Low Precision Use of expired components e Check the expiration date listed before use 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 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 a c oo n lt E e wn 2 a E mo w E o w Q x c 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 c
9. ries 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 0 781 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 100x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed FN Standard Curve OD4s0 nm 0 1 suil Le 4 1 0 10 0 100 0 FN ug ml Performance Characteristics e The minimum detectable dose of FN as calculated by 25D from the mean of a zero standard was established to be 0 7 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 25 pg ml Recovery 86 111 Average Recovery 97 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 50 104 106 1 100 99 95 1 200 96 94 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 10 Mouse None Rat None Swine None Tr
10. tive enzyme immunoassay technique that measures human FN in less than 3 hours A polyclonal antibody specific for FN has been pre coated onto a 96 well microplate with removable strips FN in standards and samples is competed with a biotinylated FN 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 e Human FN Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against FN 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 FN Standard Human FN in a buffered protein base 25 ug lyophilized e Biotinyl
11. yssaypro AssayMax Human Fibronectin 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 10 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 Fibronectin ELISA Kit Catalog No EF1045 1 Sample insert for reference use only Introduction Fibronectin FN is a major component of blood plasma the extracellular matrix and is a specific ligand for several integrin adhesion receptors 1 FN plays an important role not only in cell adhesion 2 and wound healing 3 but also in embryogenesis 4 and hematopoiesis 5 Principle of the Assay The AssayMax Human Fibronectin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of fibronectin in human plasma and serum samples This assay employs a quantitative competi
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