Human Tissue Factor Activity Kit
Contents
1. DA ssAYPRO AssaySense Human Tissue Factor Chromogenic Activity 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 70 ul AssayMix and add 10 ul of Standard Sample per well Incubate at 37 C for 30 minutes Step 2 Add 20 ul of Factor Xa Substrate per well Step 3 Read the absorbance at 405 nm at zero minutes Read at 405 nm every 5 minutes for 25 minutes Symbol Key BE Consult instructions for use Assay Template 12 11 10 Human Tissue Factor TF Chromogenic Activity Kit Two Steps Lipoprotein Catalog No CT1002b Sample insert for reference use only Introduction The transmembrane protein tissue factor TF is the physiologic trigger of coagulation in normal hemostasis TF binds and allosterically activates factor VII FVII The TF FVlla complex cleaves factor IX and X leading to thrombin generation 1 TF markedly enhances the ability of FVlla to cleave both macromolecule and small peptidyl substrates 2 3 Inducible expression of TF in a variety of pathological conditions including gram negative sepsis and acute coronary syndromes is associated with thrombosis 4 52. TF also plays important roles in vasculogenesis metastasis and angiogenesis 6 8 Principle of Assay The AssaySense Human Tissue Factor Chromogenic Activity Kit is developed to determine human TF chromogenic activity in plasma serum urine tissue and cell culture samples The assay measures the ability of lipoprotein TF FVIla to activate factor X FX to factor Xa The amidolytic activity of the TF FVIla complex is quantitated by the amount of FXa produced using a highly specific FXa substrate releasing a yellow para nitroaniline pNA chromophore The change in absorbance of the pNA at 405 nm is directly proportional to the TF enzymatic activity Caution and Warning e This product is for Research Use Only and is not intended for use in diagnostic procedures e Prepare all reagents 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 The kit should not be used beyond the expiration date Reagents The activity assay kit contains sufficient reagents to perform 100 tests using microplate method e Microplate One 96 well polystyrene microplate 12 strips of 8 wells 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 Sample Diluent 11 ml e Assay Diluent 20 ml e rhTF S
3. egression analysis of the 4 parameter curve e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Tissue Factor Chromogenic Activity Standard Curve 1 05 OD at 405 nm Ol SS 1 10 100 1000 TF pM Performance Characteristics The minimum detectable dose of TF is typically 7 5 pM This assay recognizes both natural and recombinant human TF References 1 2 3 4 5 6 7 8 Ruf W and Edgington T S 1994 FASEB J 8 385 Neuenschwander P F et al 1993 Thromb Haemost 70 970 Morrissey J H 1995 Thromb Haemost 74 185 Fuster V et al 1996 Haemostasis 26 269 Leatham E et al 1995 Br Heart J 73 10 Carmeliet P et al 1996 Nature 383 73 Ruf W and Mueller B M 1996 Curr Opin Hematol 3 379 Zhang Y etal 1994 J Clin Invest 94 1320 Version 7 2R www assaypro com e mail Support assaypro com
4. g for 10 minutes remove serum and assay Depending on application needs user should determine proper dilutions If necessary dilute samples using Sample Diluent within the range of 1x 5x 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 and assay Store samples 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 and assay The samples can be stored at 20 C or below for up to 3 months e Tissue Extract tissue samples using 50 mM Tris buffered saline pH 8 0 with 1 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant and measure the protein concentration Dilute the tissue extract 1 4 into Sample Diluent and assay Freeze the remaining extract at 20 C 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 A 4ulsample 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 o
5. ng all reagents to room temperature before use The assay is performed at 37 C for chromogenic activity assay Seal the plate with sealing tape at each step e Remove excess microplate strips from the plate frame e Freshly prepare the desired volume of the AssayMix by combining the following reagents according to the assay numbers n plus one AssayMix Reagent n 1 Assay Diluent 50 ul Human FVII 10 ul Human FX 10 pl e Add 70 ul of the above AssayMix to each well of the 96 well plate e Add 10 ul of rhTF Standard and sample per well of the 96 well plate Tap plate gently to mix wells e Incubate at 37 C for 30 minutes e Add 20 ul of FXa Substrate to each well and mix gently Read the absorbance at 405 nm at zero minutes for background O D e Seal the plate with sealing tape and incubate at 37 C and read the absorbance at 405 nm every 5 minutes for 25 minutes Ze TF Standard or Sample 10 pl 37 C for 30 minutes FXa Substrate 20 ul Read the absorbance at 405 nm at zero minutes for background O D Incubate at 37 C Read the absorbance at 405 nm every 5 minutes for 25 minutes Data Analysis e Calculate the mean value of the duplicate or triplicate for each standard and sample e _ To generate standard curve from the optimal reaction time plot the graph using the standard concentrations on the x axis and the corresponding mean 405 nm absorbance on the y axis The best fit line can be determined by r
6. r 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 Standard Curve Reconstitute the rhTF Standard 1500 pM with 3 ml of reagent grade water to generate a 500 pM 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 500 pM 1 2 with Sample Diluent to produce 250 125 62 5 31 25 15 63 and 7 81 pM solutions Sample Diluent serves as the zero standard 0 pM Any remaining solution should be frozen at 20 C and used within 30 days Standard rhTF Point pM 1 part Standard 500 pM 1 part Sample Diluent 250 0 1 part P1 1 part Sample Diluent 125 0 1 part P2 1 part Sample Diluent 62 50 Pa Apart P3 1partsampleDiluent_ 3125 Ps ___1partPS 1partSampleDiluenn 7 810 e Human FVII Add 1 2 ml reagent grade water e Human FX Add 1 2 ml reagent grade water e _ FXa Substrate Add 1 1 ml reagent grade water Assay Procedure e Prepare all reagents standard solution and samples as instructed Bri
7. tandard Lipoprotein 1 vial recombinant human TF lipoprotein e Human FVII 1 vial e Human FX 1 vial e FXaSubstrate 2 vials Storage Condition e Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store Standard Factor VII Factor X and FXa Substrate at 20 C e Store Microplate Sample Diluent and Assay Diluent at 2 8 C e Unused microplate wells may be returned to the foil pouch and resealed May be stored for up to 30 days in a vacuum desiccator e Opened diluent may be stored for up to 30 days at 2 8 C Other Supplies Required e Microplate reader capable of measuring absorbance at 405 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water e Incubator 37 C 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 use supernatants and assay Depending on application needs user should determine proper dilutions If necessary dilute samples using Sample Diluent within the range of 1x 5x The undiluted samples can be stored at 20 C or below for upto 3 months Avoid repeated freeze thaw cycles EDTA or Heparin 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
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