Ensuring that appropriate controls are in place assists users in accurately distinguishing between true positive results and potentially false positive results. Positive and negative controls are also beneficial where there is a need to troubleshoot a protocol. Within this article, we examine different types of control samples that when running an ELISA.
In order to implement a positive control, use either an endogenous solution sample that is known to contain the target protein, or a peptide or purified protein that is known to contain the immunogen sequence of the primary antibody.
Should a positive result be received from the positive control, even where the samples are negative, this illustrates that the procedure is functioning and optimized. Additionally, it shows that any negative results are valid.
Where possible, it is recommended to check the antibody datasheet for a positive control suggestion. Where there is no control suggested, the following steps offer a good starting point:
- Explore Abreviews® for mentions of the antibody. Cells, tissues or lysates with previous positive uses from reviewers can be considered a sufficient positive control.
- Explore any Omnigene or Swiss-Prot database links present on the datasheet. These specialist databases often contain lists of tissues that express a particular protein and so can also work as sufficient positive controls.
- Look at the GeneCards entry for the protein in question. These records will generally provide relative levels of expression in a range of tissues.
- Should none of these approaches prove to be suitable, it is recommended to undertake a brief literature search on PubMed for information on which cells and tissues express the protein in question.
A negative control is a sample that does not express the protein that the user is detecting. This type of control is used to check false positive results and non-specific bindings. Ideally, each plate used should include a negative control sample to ensure the results are valid.
A standard is a sample that contains a known concentration of the target protein, and is used to generate a standard curve. The image below shows a standard curve from Abcam’s Murine IL6 ELISA kit, showing a concentration ranging from 15.6 to 500 µg/mL. Where a poor standard curve occurs, it most likely means that the antibody does not bind well or does not capture the protein standard. The R2 value of the trend line should ideally be greater than 0.99.
Standard in Sample Matrix (Spike Control) Control
When using an ELISA to test serum samples, include a standard in normal diluent buffer, but it is also recommended to include a standard diluted in serum from the species being tested.
These two standards can be compared to make sure that other proteins present in the serum do not affect the standard curve, and this additional measure is known as a spike control. A spike control can illustrate that a target protein is recoverable after it has been spiked into a matrix, and acceptable results in these tests are generally with the region of 80 to 120%.
Endogenous Positive Control
An endogenous positive control is recommended when testing a recombinant protein sample. In fact, this should be a critical component of the experiment.
Detection of antibodies within recombinant proteins comes with a range of inherent difficulties. Folding of the recombinant protein could be different from its endogenous native form, which could prevent the antibody from accessing the epitope. Folding issues like this are especially common with tagged proteins, so place tags on the N or C-terminal end of the recombinant protein.
Most crucially, it is essential to make sure that the recombinant protein includes the appropriate antibody’s immunogen sequence. Due to this, an endogenous positive control is vital when validating results as well as highlighting how well the reagents (such as antibodies) and the procedure itself is working.
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