SAA ELISA 试剂盒

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 1 hour.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 15 minutes.
• Step 5. 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 to produce a 1x solution. Store for up to 30 days at 2-8 °C. Human Serum Amyloid A Standard: Reconsti tute the Human Serum Amyloid A Standard (2 μg) with 0.5 mL of Standard Diluent to generate a 4 μg/mL standard s tock solution. Allow the vial to sit for 10 minutes with gentle agi tation prior to making dilutions. Prepare duplicate or triplicate s tandard points by serially diluting from the standard stock solution (4 μg/mL) 2-fold with equal volume of MIX Diluent to produce 2, 1, 0.5, 0.25, and 0.125 μg/mL solutions. MIX Diluent serves as the zero s tandard (0 μg/mL). Aliquot remaining s tock solution to limit repeated freeze-thaw cycles . This solution should be s tored at -20 °C and used within 48 hours. Standard Point Dilution [SAA] (μg/mL) [SAA] (mIU/mL) P1 1 part Standard + 1 part MIX Diluent 2.0 1.92 P2 1 part P1 + 1 part MIX Diluent 1.0 0.96 P3 1 part P2 + 1 part MIX Diluent 0.5 0.48 P4 1 part P3 + 1 part MIX Diluent 0.25 0.24 P5 1 part P4 + 1 part MIX Diluent 0.125 0.12 P6 MIX Diluent 0.0 0.0 Biotinylated Human Serum Amyloid A Antibody (50x): Spin down the antibody briefly and dilute the desi red amount of the antibody 50-fold with MIX Diluent to produce a 1x solution. The undiluted antibody should be s tored 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 20-fold with reagent grade water to produce a 1x solution. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desi red amount of the conjugate 100-fold with MIX Diluent to produce a 1x solution. The undiluted conjugate should be s tore d at -20 °C. 5

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 s trips 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 s tore in a vacuum desiccator. Add 50 l of Human Serum Amyloid A Standard or sample 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 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 , hi t 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 , hi t 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Serum Amyloid A Antibody 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 1 hour. Wash the microplate as described above . Add 50 l of SP 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 15 minutes or until 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 a t high concentration points after s topping the reaction for about 10 minutes, which will reduce the readings . 6

• 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.