Factor XI ELISA 试剂盒

Freshly dilute all reagents and bring all reagents to room temperature before use. EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C. Human Factor XI Standard: Reconstitute the 50 ng (6.2 mU) of Human Factor XI Standard with 1 mL of EIA Diluent to generate a 50 ng/mL (6.2 mU/mL) standard 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 solution (50 ng/mL) 1:2 with EIA Diluent to produce standard solution of 25, 12.5, 6.25, 3.125, and 1.562 ng/mL solutions. EIA Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20 °C and used within 5 days. 4 Standard Point Dilution [FXI] (ng/mL) [FXI] (mU/mL) P1 Standard (50 ng/mL) 50.00 6.200 P2 1 part P1 + 1 part EIA Diluent 25.00 3.100 P3 1 part P2 + 1 part EIA Diluent 12.50 1.550 P4 1 part P3 + 1 part EIA Diluent 6.250 0.775 P5 1 part P4 + 1 part EIA Diluent 3.125 0.388 P6 1 part P5 + 1 part EIA Diluent 1.562 0.194 P7 EIA Diluent 0.000 0.000 Biotinylated Human Factor XI Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with EIA 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 EIA Diluent. Any remaining solution should be frozen at -20 °C.

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 securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human Factor XI 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 Factor XI Antibody to each well and incubate for 1 hour. Wash the microplate as described above. 5 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 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 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.

• 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.