AssayMaxTM Human Fibrinogen ELISA Kit
Contents
1. 1 part MixDiluent 10 Ps part PS a part MX Diluent 25 es MX went T e Biotinylated Human Fibrinogen Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 50 fold with MIX Diluent to produce a 1x solution The undiluted antibody should be stored 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 20 fold with reagent grade water to produce a 1x solution e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100 fold with MIX Diluent to produce a 1x solution The undiluted conjugate should be stored 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 Fibrinogen Standard or sample to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash the microplate manually or automatically usi2. actor XIlla to form an insoluble network FBG also binds to the platelet glycoprotein IIb Illa receptor to form bridges between platelets thus facilitating aggregation 2 Elevated plasma FBG has been identified as an independent risk factor for coronary atherosclerosis and ischemic heart disease 3 4 Individuals with congenital absence of FBG termed afibrinogenemia have prolonged bleeding times Principle of the Assay The AssayMax Human Fibrinogen ELISA Enzyme Linked Immunosorbent Assay Kit is designed for detection of FBG in human milk urine saliva CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human FBG in approximately 4 hours A polyclonal antibody specific for human FBG has been pre coated onto a 96 well microplate with removable strips FBG in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for human FBG which is recognized by a streptavidin peroxidase SP 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 intended for use in diagnostic procedures Prepare all reagents diluent buffer wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assay e Prepare
3. 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 Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents The Stop Solution is an acidic solution The kit should not be used beyond the expiration date Reagents Human Fibrinogen Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human FBG Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Human Fibrinogen Standard Human FBG in a buffered protein base 64 ng lyophilized Biotinylated Human Fibrinogen Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against human FBG 120 tl 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 2 bottles SP Conjugate 100x A 100 fold concentrate 80 yl Chromogen Substrate 1x A stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution 1x A 0 5 N hydrochloric acid solution 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 an
4. d Biotinylated Antibody 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 Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required Microplate reader capable of measuring 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 Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes A 30 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted 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 pot Centrifuge samples at 800 x g for 10 minutes The sample is suggested for use at 1x however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes An 80 fold sample dilution is suggested into MIX Diluent however u
5. ds e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution Contamination of reagents Contents of wells evaporate 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 e A new tip must be used for each addition of different samples or reagents during the assay procedure 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 10 e Thoroughly agitate the lyophilized components after Insufficient mixing of ae 5 reconstitution reagent dilutions e Thoroughly mix dilutions References 1 2 3 4 Doolittle RF 1984 Annu Rev Biochem 53 195 Handley DA Hughes TE 1997 Thromb Res 87 1 Handa K et al 1989 Atherosclerosis 77 209 Mannucci PM Mari D 1993 Fibrinolysis 3 51 Version 4 8 www assaypro com e mail support assaypro com
6. l 4 1 10 100 FBG ng ml Performance Characteristics e The minimum detectable dose of human FBG as calculated by 2SD from the mean of a zero standard was established to be 0 7 ng ml e Intra assay precision was determined by testing three reference control samples twenty times in one assay e Inter assay precision was determined by testing three reference control samples in twenty assays Intra Assay Precision Inter Assay Precision CV 4 5 4 2 4 9 9 9 9 7 10 1 Average o CV 4 5 9 9 Recovery Standard Added Value 2 5 40 ng ml Recovery 88 109 Average Recovery 96 Linearity e Milk samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Milk 15x 94 Cross Reactivity Species Cross Reactivity Bovine None Mouse None Rat None Swine None Monkey lt 5 Canine None Rabbit None Troubleshooting Causes Course of Action Low Precision Use of improper components e Check the expiration date listed before use e Do not interchange components from different lots Improper wash step Splashing of reagents while loading wells e Check that the correct wash buffer is being used e Check that all wells are empty after aspiration e Check that the microplate washer is dispensing properl
7. late 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 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 laboratories may be caused by technique differences Standard Point Average OD P1 2 052 P2 40 TA 1 687 P3 20 ae 1 095 P4 10 a 0 655 P5 5 0 os 0 387 P6 25 set 0 249 P7 1 25 o 0 175 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human FBG Standard Curve nm r OD 450 0 1 sasl cad 4 tr r
8. ng a microplate washer Invert the plate and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If washing manually wash five times with 200 ul of Wash Buffer per well Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If using a microplate washer wash six times with 300 ul of Wash Buffer per well invert the plate and hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Human Fibrinogen Antibody to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of SP Conjugate to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Cover wells with a sealing tape 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 to each well Gently tap plate to thoroughly coat the wells Break any bubbles that may have formed Incubate for 12 minutes or until the optimal blue color density develops e Add 50 ul of Stop Solution to each well The color will change from blue to yellow Gently tap plate to ensure thorough mixing Break any bubbles that may have formed e Read the absorbance on a microp
9. ser should determine optimal dilution factor depending on application needs 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 A 200 fold sample dilution is suggested into MIX Diluent however user should determine optimal dilution factor depending on application needs The undiluted samples can be stored at 80 C for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatant Centrifuge cell culture media at 1500 rpm for 10 minutes at 4 C to remove debris and collect supernatant Samples can be stored at 80 C Avoid repeated freeze thaw cycles Applicable samples may also include biofluids cell culture and tissue homogenates If necessary user should determine optimal dilution factor depending on application needs Refer to Dilution Guidelines for further instruction Guidelines for Dilutions of 100 fold or Greater for reference only please follow the insert for specific dilution suggested 100x 10000x A 4ulsample 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 1000x 100000x 4 ul sample 396 ul buffer 100x 24
10. 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 10 fold with reagent grade water to produce a 1x solution Store for up to 30 days at 2 8 C e Human Fibrinogen Standard Reconstitute the Human Fibrinogen Standard 64 ng with 0 8 ml of MIX Diluent to generate an 80 ng ml standard stock solution Allow the vial to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting from the standard stock solution 80 ng ml 2 fold with equal volume of MIX Diluent to produce 40 20 10 5 2 5 and 1 25 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining stock solution should be stored at 20 C and used within 30 days Avoid repeated freeze thaw cycles Standard face FBG Point Dilution ng ml P1 1 part Standard 80 ng ml 80 1 part P1 1 part MIX Diluent 40 1 part P2 1 part MIX Diluent a a part P3
11. y e If washing by pipette check for proper pipetting technique 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 Unexpectedly Low or High Signal Intensity 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 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 perio
12. yssarpro AssayMax Human 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 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 12 minutes Step 5 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 Human Fibrinogen ELISA Kit Catalog No EF2040 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 Elevated and decreased levels have clinical significance Upon cleavage by thrombin in the initial stages of coagulation activation FBG self assembles to yield a fibrin clot matrix that subsequently is cross linked by f
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