AssayMax Swine Prothrombin ELISA Kit
Contents
1. calculated by 2SD from the mean of a zero standard was established to be 0 30 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 Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 1 100 ng ml Recovery 91 110 Average Recovery 96 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 6000 91 92 1 12000 96 96 1 24000 95 89 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey None Mouse None Rat None Rabbit None Swine 100 Human None Troubleshooting 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 an2. DA assarbro AssayMax Swine Prothrombin 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 15 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key 3 Consult instructions for use Assay Template 12 11 10 Swine Prothrombin ELISA Kit Catalog No EPP3022 1 Sample insert for reference use only Introduction Prothrombin is also known as Factor Il The conversion of Factor X to Xa changes prothrombin into its active form thrombin which then accelerates the formation of fibrin The level of the plasma prothrombin in the circulating blood decreases during its passage through the pulmonary capillaries 1 The bleeding tendency in acute chloroform intoxication is due to deficiency in both plasma fibrinogen and plasma prothrombin 2 It has been shown t
3. andard 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 400 ng ml 1 4 with EIA Diluent to produce 100 25 6 25 1 563 and 0 391 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 30 days Standard Je Swine Prothrombin 2 Dilution Point ng ml P1 1 part Standard 400 ng ml 400 0 1 part P1 3 parts EIA Diluent 100 0 1 part P2 3 parts EIA Diluent 25 00 Pa 1partP3 3partsFlADiluent 6250 Pe 1partPS 3partsFlADiluent os e Biotinylated Swine Prothrombin Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 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 str
4. ax Mouse Prothrombin ELISA Kit Plasma Serum Urine and Cell Culture samples www assaypro com e e mail Support assaypro com
5. d 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 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 periods e Consult the provided procedure for correct incubation tim
6. e 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 Insufficient mixing of eis oe 5 reconstitution reagent dilutions e Thoroughly mix dilutions References 1 William DEW Andrus et al 1940 Science 91 2350 48 50 2 H P Smith et al 1937 The Journal of Experimental Medicine 66 801 811 3 Zipser BD et al 2006 Neurobiol Aging June 15 Version 1 4R Related Products e _ ET4010 1 AssayMax Human Thrombin ELISA Kit Cell Culture samples EP3022 1 AssayMax Human Prothrombin ELISA Kit Plasma Milk Urine and Cell Culture samples EMP3022 1 AssayM
7. e 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 swine plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x g for 10 minutes Samples are recommended for use at 1 12000 into EIA Diluent and assay 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 Heparin and EDTA 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 Samples are recommended for use at 1 12000 into EIA Diluent and assay 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 Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 mi
8. hat Prothrombin is localized within the wall and neuropil surrounding microvessels in certain disorders 3 Principle of the Assay The AssayMax Swine Prothrombin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of swine prothrombin in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures swine prothrombin in less than 4 hours A monoclonal antibody specific for swine prothrombin has been pre coated onto a 96 well microplate with removable strips Prothrombin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for swine prothrombin 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 determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e T
9. he stop solution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Swine Prothrombin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a monoclonal antibody against swine prothrombin 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 _ Swine Prothrombin Standard Swine prothrombin in a buffered protein base 800 ng lyophilized e Biotinylated Swine Prothrombin Antibody 100x A 100 fold concentrated biotinylated polyclonal antibody against swine prothrombin 60 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 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 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 th
10. hose 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 Calculate the mean value of the duplicate or triplicate readings for each standard and sample 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 Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data 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 400 0 P2 100 0 P3 25 00 P4 6 250 PS 1 563 P6 0 391 P7 0 000 Sample Swine Plasma 12000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Swine Prothormbin Standard Curve i OD 450 nm 10 i 10 10 10 Prothrombin ng ml Sa Performance Characteristics e The minimum detectable dose of swine prothrombin as
11. ips 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 Swine Prothrombin Standard or sample per well cover wells 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 Add 50 ul of Biotinylated Swine Prothrombin Antibody to each well and incubate for 1 hour Wash the microplate as described above 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 Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 15 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 Add 50 ul of Stop Solution to each well The color will change from blue to yellow 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 t
12. nutes to remove debris Collect supernatants and assay Store samples at 20 C or below 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 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 Standard Curve Reconstitute the 800 ng of Swine Prothrombin Standard with 2 ml of EIA Diluent to generate a 400 ng ml standard stock solution Allow the st
Download Pdf Manuals
Related Search