There are five main types of control samples you can use when running an ELISA to ensure the accuracy of your results. These controls help distinguish true positive results from false positives or negatives and are also useful for troubleshooting.
When running an ELISA, it’s advisable to use these five types of control samples:
Positive Control contains the protein or peptide you are detecting. This can either be an endogenous sample known to express the protein or a purified protein with the immunogen sequence for the antibody you are using. A positive result from the positive control is an indication that the procedure is optimized and working correctly. It validates that any negative results are genuine.
Negative Control is a sample that’s known to not express the target protein you are detecting. This helps identify any non-specific binding and ensures there are no false positives. To validate the overall results, every plate used in the experiment should contain a negative control sample.
Standard Control contains a known concentration of the target protein and is used to generate a standard curve. This helps determine the concentration of the protein in your test samples. A good standard curve will have an R2 value > 0.99, indicating that the antibody binding is effective. A poor standard curve indicates that the antibody did not bind properly.
Standard in Sample Matrix (Spike Control): When testing complex samples such as serum in ELISA, it’s recommended to include a standard in normal diluent buffer as well as a standard diluted in serum from the species being tested. This allows you to compare and ensure that the sample matrix doesn’t interfere with the standard curve, and is known as a spike control. It indicates that a target protein is recoverable after spiking into a matrix. Acceptable recovery results range between 80-120%.
Endogenous Positive Control is an essential component of your experiment if you are working with recombinant proteins. There are a few challenges inherent with antibody detection of recombinant proteins. Recombinant proteins may vary from the endogenous native form in folding or they may prevent the antibody from accessing the epitope, particularly if they are tagged. Including an endogenous positive control ensures that the reagents and procedure are effective and that the protein of interest is correctly detected.