Vitamin D-Binding Protein ELISA 试剂盒

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 100 g of GC-Globulin Standard with 1 ml of EIA Diluent to generate a solution of 100 g/ml. Allow the standard to sit for 10 minutes with 2 gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (100 g/ml) 1:4 with EIA Diluent to generate 25, 6.25, 1.56, 0.391 and 0.098 g/ml solutions. EIA Diluent serves as the zero standard (0 g/ml). Any remaining solution should be frozen at -20°C. Standard Point Dilution [hGC-Globulin] (g/ml) Standard (100 g/ml) P1 100.000 P2 1 part P1 + 3 parts EIA Diluent 25.000 P3 1 part P2 + 3 parts EIA Diluent 6.250 P4 1 part P3 + 3 parts EIA Diluent 1.563 P5 1 part P4 + 3 parts EIA Diluent 0.391 P6 1 part P5 + 3 parts EIA Diluent 0.098 P7 EIA Diluent 0.000 Biotinylated GC-Globulin (1x): Dilute Biotinylated GC-Globulin with 4 ml EIA Diluent to use. Allow to sit for 10 minutes with gentle agitation prior to use. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (10x): Dilute the Wash Buffer Concentrate 1:10 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 25 µL of standard or sample per well, and immediately add 25 µL of Biotinylated GC- Globulin to each well (on top of the Standard or sample) and mix gently. 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 Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash a microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 8 minutes or till the optimal blue color density develops. Gently tap 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 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 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. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.