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Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 640 ng of human Thrombin Standard with 4 ml of EIA Diluent to generate 160 ng/ml of stock solution. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. The stock solution can be further dilute 1:8 with EIA Diluent to produce 20 ng/ml standard solution. Prepare triplicate standard points by serially diluting the Thrombin standard solution 1:2 with equal volume of EIA Diluent to produce 10, 5, 2.5, 1.25, 0.625 and 0.313 ng/ml. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [alpha Thrombin] (ng/ml) P1 Standard (20 ng/ml) 20.000 P2 1 part P1 + 1 part EIA Diluent 10.000 P3 1 part P2 + 1 part EIA Diluent 5.000 P4 1 part P3 + 1 part EIA Diluent 2.500 P5 1 part P4 + 1 part EIA Diluent 1.250 P6 1 part P5 + 1 part EIA Diluent 0.625 P7 1 part P6 + 1 part EIA Diluent 0.313 P8 EIA Diluent 0.000 2 Biotinylated Thrombin 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. Wash Buffer Concentrate (20x): Dilute the Wash Buffer 1:20 with reagent grade water. 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.
Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer. Invert the plate and decant the contents, and hit it 4-5 times on absorbent paper towel to complete remove liquid at each step. Add 50 µL of Biotinylated Thrombin Antibody to each well and incubate for one hour. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Streptavidin-Peroxidase Conjugate per well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash five times with 200 µL of Wash Buffer as above. Add 50 µL of Chromogen Substrate per well and incubate for approximately 10 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 µL 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. Please note that some unstable black particles may be generated at high optical densities to reduce the readings after stopping the reaction for about 10 minutes.
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 on the y-axis. The best-fit line can be determined by regression analysis using four-parameter or log-log logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.