What controls do I need for ELISA?

For confidence in ELISA results, it is necessary to have three experimental controls for comparison: a positive control, negative control, and a standard control. Both the positive and standard controls are known to contain the protein or peptide of interest.

The positive control is to confirm simply if the procedure is performing as intended. A positive control serves three functions: it allows for confidence in the experimental results, confirms that negative results are accurate, and assists in any protocol adjustments or optimizations that may be necessary.

The standard control is necessary for quantification of the experimental results. It contains a known quantity of the target molecule and therefore gives the information necessary to make a standard curve. The standard control can also be useful for optimization, as an unsatisfactory standard curve can be indicative of poor antibody binding or inadequate specificity for the target protein.

The negative control, as the title suggests, is known to not contain the biomolecule of interest, and adds validity to any positive results. A poorly-chosen antibody that does not bind with sufficient specificity will also contribute to a false result in the negative control. If this is the case, further troubleshooting of the experimental design is needed.

In the interest of time or resource constraints, some researchers use well-tested kits for ELISA preparation. Some selected examples are in Table 1 below. 

Table 1: Selected ELISA Assay Kits