AssayMaxTM Human GPIIb/IIIa ELISA Kit
Contents
1. Recovery Standard Added Value 2 5 20 ng ml Recovery 91 111 Average Recovery 101 Linearity e Plasma samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma 1 20 91 1 40 97 1 80 95 Cross Reactivity Species Cross Reactivity Canine None Monkey 20 Mouse 2 Rat None Swine 2 Rabbit None Bovine 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 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 pu2. 1 part P1 1 part MIX Diluent 20 00 1 part P2 1 part MIX Diluent 10 00 P4 1partP3 1 part MIX Diluent 5 000 Pe 1partP5 1 part MIX Diluent 1250 IP MXDiluent_ ooo e Biotinylated Human GPlIb llla Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 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 GPilb Illa 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 time
3. llla serves as an inducible receptor for fibrinogen fibronectin von Willebrand factor and vitronectin 2 3 The simultaneous occupancy on adjacent platelets of receptors with dimeric fibrinogen molecules leads to platelet aggregation 4 Principle of the Assay The AssayMax Human GPilb Illa ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human GPIlb Illa in platelets platelet rich plasma and cell culture lysate samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures GPIIb Illa in less than 4 hours A polyclonal antibody specific for human GPIIb IIla has been pre coated onto a 96 well microplate with removable strips GPlIb llla in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for GPIlb Illa 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 dete
4. 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 see ing reconstitution reagent dilutions e Thoroughly mix dilutions References 1 Kuhn K and Eble J 1994 Trends Cell Biol 4 256 2 Kieffer N and Phillips D R 1990 Annu Rev Cell Biol 6 329 3 Ruggeri Z M etal 1983 J Clin Invest 72 1 4 George J N etal 1990 Blood 75 1383 Version 5 8R www assaypro com e mail Support assaypro com
5. ate 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 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 e 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 Platelet Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant containing 1 uM prostaglandin E1 Centrifuge samples at 100 x g for 15 minutes to obtain platelet rich plasma To sediment the platelets the platelet rich plasma is further centrifuged at 1000 x g for 10 minutes The platelet pellet is then washed twice in Tyrode s HEPES buffer pH 7 4 containing albumin 0 5 and prostaglandin E1 1 uM The platelet is dissolved with 100 mM n octylglycoside buffer pH 7 4 in 20 mM HEPES buffered saline Dilute samples 1 40 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 e Platelet Rich Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoa
6. gulant containing 1 uM prostaglandin E1 Centrifuge samples at 100 x g for 15 minutes to obtain platelet rich plasma Dilute samples 1 40 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 e Cell Culture Lysates The cultured cells are lysed and solubilized with 15 mM octyl B D glucopyranoside at 37 C for 15 minutes Collect fresh cell lysates dilute it with MIX Diluent and assay 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 Standard Curve Reconstitute the 48 ng of Human GPlIb llla Standard with 1 2 ml of MIX Diluent to generate a 40 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 40 ng ml twofold with equal volume of MIX Diluent to produce 20 10 5 2 5 1 25 and 0 625 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 3 days Standard Point Dilution GPlIbilla ng ml P1 1 part Standard 40 ng ml 40 00
7. nctures e Check that three desiccants are inside the microplate pouch prior to sealing a ce oo 72 lt E e wn 2 a E mo w E o w Q x c gt 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 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
8. rd 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 40 00 P2 20 00 P3 10 00 P4 5 000 PS 2 500 P6 1 250 P7 0 625 P8 0 000 Sample Normal Sodium Citrate Plasma 40x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human GPIIb Illa Standard Curve OD 450 nm iil po iil pa iil pi iil 0 1 1 10 100 hGPIIb Illa ng ml Performance Characteristics e The minimum detectable dose of GPIlb Illa as calculated by 2SD from the mean of a zero standard was established to be 0 5 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
9. rmine 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 GPlIb llla Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against GPIlb IIla e Sealing Tapes Each kit contains 3 pre cut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Human GPlIb llla Standard Human platelet GPIIb llla in a buffered protein base 48 ng lyophilized e Biotinylated Human GPlIb llla Antibody 100x A 100 fold concentrated biotinylated polyclonal antibody against human GPilb Illa 60 ul 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 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 Micropl
10. s 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 Biotinylated Human GPilb Illa Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate to each 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 Add 50 ul of Chromogen Substrate per well and incubate for 15 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 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 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 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 standa
11. yssaypro AssayMax Human GPilb Illa 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 ci Consult instructions for use Assay Template 12 11 10 Human Glycoprotein IIb IIla GPIIb llla ELISA Kit Catalog No EG1060 1 Sample insert for reference use only Introduction Platelet membrane glycoprotein Ilb Illa GPIlb Illa integrin o 3 is a member of the integrin family of cell membrane receptors that play key roles in thrombus formation platelet aggregation embryogenesis and intercellular adhesion Each integrin receptor complex consists of a heavy alpha and a light beta chain associated as a calcium dependent heterodimer with a molecular mass of 140 kDa and 90 kDa respectively 1 GPlIb
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