Required controls for ELISA

Running the appropriate controls helps you to accurately separate true positive results from potentially false results. Positive and negative controls will also be useful if you ever need to troubleshoot your protocol. Here we explain the various types of control samples you should use when running an ELISA.

Use either an endogenous soluble sample known to contain the protein you are detecting or a purified protein or peptide known to contain the immunogen sequence for the antibody you are using. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.

We recommend checking the antibody datasheet, which will often provide a suggested positive control. If no control is suggested, we recommend the following: 

• Check to see if there are any product reviews for the antibody. Any tissues, cells, or lysates that have been used successfully can be considered a suitable positive control.
• Try looking at the Swiss-Prot or Omnigene database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
• Check the GeneCards entry for the protein. This will usually provide you with relative levels of expression in various tissues.
• If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.

This is a sample that you know does not express the protein you are detecting. This is to check for non-specific binding and false positive results. Each plate you use should contain a negative control sample in order to validate the results.

This is a sample that contains a known concentration of the target protein from which the standard curve can be obtained. For example, below is a typical standard curve from our Murine IL6 ELISA kit with concentration ranging from 15.6 to 500 µg/mL. A poor standard curve means the antibody didn’t bind properly or doesn’t capture the protein standard. The R² value of the trend line should be >0.99.

When testing serum samples in ELISA, include a standard in normal diluent buffer as usual. But we recommend to also include a standard diluted in serum from the species you are testing. The two can then be compared to ensure there is no effect on the standard curve from other proteins in the serum. This is known as a spike control and tells you that a target protein is recoverable after being spiked into a matrix. Acceptable results are 80–120%.

We recommend including an endogenous positive control if you are testing a recombinant protein sample. This should be an essential component of your experiment.

There are inherent difficulties with antibody detection of recombinant proteins that need to be considered. Folding of the recombinant protein may be different from the endogenous native form, and may prevent antibody access to the epitope. This is particularly the case with tagged proteins. Always ensure tags are placed on the N or C-terminal end of the recombinant protein.

Most importantly, always ensure the recombinant protein includes the immunogen sequence of the antibody you are using. An endogenous positive control is important to validate the results, as well as to indicate how well the reagents (eg antibodies) and procedure are working.