Fibronectin ELISA 试剂盒

• Step 1. Add 25 μL of Standard or Sample and 25 μL of Biotinylated Protein per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 3. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 25 minutes.
• Step 4. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

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 Concentrate 10-fold with reagent grade water. Store for up to 30 days at 2-8 °C. Human Fibronectin Standard: Reconstitute the 65 g of Human Fibronectin Standard with 1.3 mL of MIX Diluent to generate a 50 g/mL 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 from the standard stock solution (50 g/mL) 2-fold with MIX Diluent to produce 25, 12.5, 6.25, 3.125, 1.563, and 0.781 g/mL solutions. MIX Diluent serves as the zero standard (0 g/mL). Any remaining stock solution should be frozen at -20 °C and used within 30 days. Avoid repeated freeze-thaw cycles. 4 Standard Point Dilution [FN] (μg/mL) P1 1 part Standard (50 g/mL) 50 P2 1 part P1 + 1 part MIX Diluent 25 P3 1 part P2 + 1 part MIX Diluent 12.5 P4 1 part P3 + 1 part MIX Diluent 6.25 P5 1 part P4 + 1 part MIX Diluent 3.125 P6 1 part P5 + 1 part MIX Diluent 1.563 P7 1 part P6 + 1 part MIX Diluent 0.781 P8 MIX Diluent 0.0 Biotinylated Human Fibronectin Protein (1x): Reconstitute Biotinylated Human Fibronectin Protein with 3 mL of MIX Diluent to produce a stock solution. Allow the vial to sit for 10 minutes with gentle agitation prior to use. Any remaining stock solution should be frozen at -20 °C and used within 30 days. Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 20-fold with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 100-fold with MIX Diluent. The undiluted conjugate should be stored 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 25 l of Human Fibronectin Standard or sample per well and immediately add 25 l of Biotinylated Human Fibronectin Protein to each well (on top of the standard or sample). Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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. 5 Add 50 l of Streptavidin-Peroxidase Conjugate to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Cover wells with a sealing tape 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 to each well. Gently tap plate to thoroughly coat the wells. Break any bubbles that may have formed. Incubate for 25 minutes or till the optimal blue color density develops. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Gently tap plate to ensure thorough mixing. Break any bubbles that may have formed. 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 low 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.