Total tPA ELISA Kit
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- Target
- Total tPA
- Reactivity
- Human
- Detection Method
- Colorimetric
- Method Type
- Sandwich ELISA
- Detection Range
- 0.313-10 ng/mL
- Minimum Detection Limit
- 0.313 ng/mL
- Application
- ELISA
- Purpose
- The AssayMax™ Human Total tPA ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human tPA in plasma, serum, milk, cell culture, and tissue extract samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures total tPA in less than 4 hours. A polyclonal antibody specific for tPA has been pre-coated onto a 96-well microplate with removable strips. Human tPA in standards and samples is sandwiched by the immobilized antibody and a biotinylated antibody specific for tPA, 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.
- Brand
- AssayMax™
- Sample Type
- Cell Culture Cells, Milk, Plasma, Serum, Tissue Lysate
- Analytical Method
- Quantitative
- Components
- Human tPA Microplate: A 96 well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against tPA. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human tPA Standard: Human tPA in a buffered protein base (10 ng, lyophilized). Biotinylated Human tPA Antibody (50x): A 50-fold concentrated biotinylated antibody against human tPA (140 l). 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). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).
- Material not included
- Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)
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- Plate
- Pre-coated
- Protocol
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- Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
- Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
- Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
- Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 12 minutes.
- Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.
- Reagent Preparation
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Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute MIX Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C.
- Sample Collection
- 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. Dilute samples 1:2 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 or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes. Dilute samples 1:2 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. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes and assay. Samples can be stored at -20 °C or below. Avoid repeated freeze-thaw cycles. Tissue Extracts: Extract tissue samples with 50 mM phosphate-buffered saline ( pH 7.4) containing 1 % Triton X-100 and centrifuge at 14000 x g for 20 minutes. Collect the supernatant, measure the protein concentration, and assay. The samples can be stored at -20 °C or below for up to 3 months. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:2 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 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000 fold dilution Assuming the needed volume is less than or equal to 400 μL. 1:1000 1:100000 A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000 fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000 fold dilution Assuming the needed volume is less than or equal to 240 μL. 4
- Assay Procedure
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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). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch 5 securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human tPA Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l 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 l 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 l of Biotinylated Human tPA Antibody to each well and incubate for 1 hour. Wash the microplate as described above. Add 50 l 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 l of Chromogen Substrate per well and incubate for 12 minutes or till the optimal blue color density develops. Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 l 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.
- Calculation of Results
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- 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.
- Restrictions
- For Research Use only
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- Handling Advice
- 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. 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. 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. 2
- Storage
- 4 °C,-20 °C
- Storage Comment
- Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and 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 desiccants and resealed. May be stored for up to 30 days in a vacuum desiccator. Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.
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- Target
- Total tPA
- Alternative Name
- tPA (Total tPA)
- Background
- Tissue-type plasminogen activator (tPA) is a serine protease that converts the zymogen plasminogen into the active serine protease plasmin, the primary enzyme responsible for the removal of fibrin deposits (1). tPA is a 68 kDa glycoprotein that is synthesized by endothelial cells in normal blood vessels and displays relatively high affinity for fibrin, suggesting that it functions predominately in physiological thrombolysis in vivo (2).
- Gene ID
- 5327
- UniProt
- P00750
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