ELISA protocols vary by subtypes, but share basis
There are five types of ELISA, thus, about ELISA protocol, a few differences exist amid indirect ELISA protocol, direct ELISA protocol, sandwich ELISA protocol, competitive ELISA protocol and ELISPOT protocol. However, the main ELISA principle and lots of procedures are the same. General ELISA protocol includes plate preparation, assay procedure and calculation of results.
(ELISA Protocol) Plate Preparation
1.Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.
2.Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
3.Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
4.Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
(ELISA Protocol) Assay Procedure
1.Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.
2.Repeat the aspiration/wash as in step 2 of plate preparation.
3.Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.
4.Repeat the aspiration/wash as in step 2 of plate preparation.
5.Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light.
6.Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
7.Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
(ELISA Protocol) Calculation of Results
1.Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.
2.Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.
3.To determine the concentration of the unknowns, find the unknowns' mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
4.Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.