产品名称：Thrombin-AntithrombinComplex(TAT) ELISA Kit

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 the MIx Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. TAT Complexes Standard: Reconstitute the 8 ng of mouse TAT Complexes Standard with 1 ml of MIx Diluent to generate a standard solution of 8 ng/ml. 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 solution 1:2 with MIx Diluent to produce 4, 2, 1, 0.5, 0.25 and 0.125 ng/ml. MIx Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be frozen at -20°C for up to one week. Standard Point Dilution [TAT] (ng/ml) P1 Standard Stock (8 ng/ml) 8.000 P2 1 part P1 + 1 part MIx Diluent 4.000 P3 1 part P2 + 1 part MIx Diluent 2.000 P4 1 part P3 + 1 part MIx Diluent 1.000 P5 1 part P4 + 1 part MIx Diluent 0.500 P6 1 part P5 + 1 part MIx Diluent 0.250 P7 1 part P6 + 1 part MIx Diluent 0.125 P8 MIx Dilutent 0.000 Biotinylated Antithrombin Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with MIx Diluent. Any remaining solution should be frozen at -20°C. 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. 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.

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 manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated TAT Antibody to each well and incubate for two hours. Wash the microplate as described 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. 3 Wash the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 20 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 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. 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.

Calculate the mean value of the 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 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. 3 Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.