Complement C8 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 EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. 4
Standard Curve: Reconstitute the 40 ng of human C8 standard with 1 mL of EIA Diluent to generate a stock standard solution of 40 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 C8 standard solution 1:2 with equal volume of EIA Diluent to produce 20, 10, 5, 2.5, 1.25, and 0.625 ng/mL. EIA Diluent serves as the zero standard (0 ng/mL). Any remaining solution should be frozen at -20°C and used within the next 30 days.
Biotinylated C8 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 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.

Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. Dilute samples 1:10000 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant).
Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes. Remove serum and dilute samples 1:10000 into EIA Diluent. The undiluted samples can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. The samples can be stored at -20°C or below. Avoid repeated freeze-thaw cycles.
Saliva: Collect saliva using sample tube. Centrifuge samples at 800 x g for 10 minutes and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.
Milk: Centrifuge samples at 800 x g for 10 minutes and assay. Dilute samples 1:20 into EIA Diluent. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles.

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 desiccants 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 2 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 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, decanting the contents, hit 4-5 times on absorbent paper towel to completely remove the liquid.
Add 50 µL of C8 Biotin to each well and incubate for 1 hour.
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.
Wash the microplate as described above.
Add 50 µL of Chromogen Substrate per well and incubate for approximately 15 minutes or till the optimal blue 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 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.