ELISA procedure is an immunoassay technique which provides an instrumental tool to detect or quantify the concentration of a specific analyte. The standard format of an ELISA assay is a 96 microwell plate (usually in an 8 row by 12 column). Each microwell is pre-treated to generate a response, which in majority of cases involves a colour change within the solution present in the microwell. A spectrophotometer is used to detect the quantity of light that passes through this solution. The wells change from clear (no reaction) to dark blue (100% reaction), has the colour gets darker this is detected by a change in the optical density reading recorded on the spectrophotometer. The concentration of the unknown analyte is compared against a standard curve, that is constructed using standard preparation of known concentrations.
Types of ELISA data output
There are essentially 3 different types of data output that can be obtained.
- Qualitative ELISA: This assay provides a simple positive or a negative result, it can only confirm if any antigen is present by comparing to a blank well with no antigen or an unrelated control antigen.
- Semi-Quantitative ELISA: This assay compares the relative amounts of the antigen present within the sample being tested. The intensity of the signal detected will directly correspond to the amount of antigen present. However, with this procedure it is not possible to calculate the exact concentration since there is no standard protein present within the ELISA kit.
- Quantitative ELISA: This assay calculates the exact amount of the antigen present within the sample. A standard curve is constructed by a series of serial dilutions using standard proteins of known concentrations. The unknown concentration of the antigen can be interpolated from the standard curve to calculate the amount of antigen present within the sample. This procedure is most frequently used to report ELISA data.
ELISA assay standard curve
The concentration of the unknown antigen in the sample that is been tested is calculated quantitively using a standard or calibration curve. The standard curve is plotted using the known concentrations of the standard reference antigens against the optical density for each concentration (usually at 450nm using a plate reader).
Majority of plate readers will usually incorporate a software that will automate the data analysis and curve fitting process. The calculation of the unknown antigen is carried out through extrapolating the linear part of the standard curve. It is highly recommended when performing an ELISA assay to run samples either in duplicates or triplicates to ensure good practice statistical validation of the results. The standard deviation (SD) and the coefficient of variation (CV) can be calculated to assess the precision of pipetting, for best practice CV values should be kept below 20%.
Also, it is advisable to include positive controls (samples with known analyte quantity or presence of your analyte) and negative controls (samples with no analyte quantity or presence) during the ELISA plate setting up process.
Curve fitting software
- Semi-log plot: this uses the log of the concentration against the microplate readout. A typical sigmoid curve is generated which results in distributing the data points more evenly. The method is useful in counteracting the lower end compression that is due to the linear plots.
- Linear plot: a curve is plotted with the antigen concentrations on one axis against the microplate readings on the other axis. For majority of the cases R2 values greater than 0.99 are indicative of a good fit curve. Unfortunately, linear plots are found to compress the data points especially at the lower end of the curve and this can lead to the resolution being lower.
- Log/log plot: this is common for low to medium concentration ranges since it offers a good linearity over this range. However, if the concentration is increased to the higher range then the linearity is found to be lost.
- 4PL or 5PL curves (4- or 5-parameter logistics): these are highly sophisticated procedures that require calculations that are more complex. The 4PL method is based on the symmetry being around the infection point whereas the 5PL method considers asymmetry, for majority of immunoassays 5PL is often a much better fit.
Spike recovery is useful in helping to determine if there are any components present within the sample that are likely to interfere with the antibody to antigen binding process. This procedure is carried out by spiking the sample matrix using a known concentration of a recombinant protein. The standard ELISA assay is carried out and the concentration of the protein is extrapolated from the standard curve. The recovery of the protein is expressed as a percentage, generally, a value below 80% will indicate presence of components within the matrix that are interfering with the assay. In this situation we would recommend using a different ELISA kit.
Coefficient of variation (CV)
Coefficient of variation is important to determine any inaccuracies or inconsistency within the data or results obtained. This is usually expressed as the percentage of variance against the mean. The expected inconsistency or inaccuracies become greater with an increase in the variance. Some of the common factors that can contribute to high coefficient of variations (CV) include sample contamination, variation in temperature, pipetting errors, insufficient washing and wells drying out.