Recovery is a fundamental biophysical property in the immunoassay developer community. As we expand assay content gold standards such as ELISA, or examine new platforms, a common goal is that the assay system measure as much of the sample as possible without artifacts or interference. A well-designed assay system enables the end user to generate an analyte standard curve that closely emulates antibody-antigen binding properties that naturally take place during incubations of the unknown sample and the assay reagents. Below, my colleague Jamie Boden, a technical writer for our Immunoassay Systems, explains the concept of Spike-and-Recovery and how to ensure success during your next immunoassay run.
Developing your own ELISA can be quite a task! There are many things to consider when building your own immunoassay – antibodies, buffers, diluents, plates, and more. Whether you are choosing to develop your own assay as a cost savings or because you already have a reliable antibody pair, you must make sure you are detecting all of your protein of interest. It would be wasted effort if your assay measurements only detected some (but not all) of the protein present in your sample.
The best way to ensure you are measuring the true concentration is to run a spike-and-recovery experiment. Spike-and-recovery testing determines if your standard diluent and sample matrix (plasma, serum, etc.) are interfering with analyte binding to the capture and detection antibodies used in your immunoassay. To test whether you have captured all the protein you “spike,” a known concentration of protein into the diluent (just as in a standard curve), as well as the matrix to see how much of that concentration you “recover” upon measurement. Ideally, your results should be as close to 100% recovery as possible.
100% recovery means there is no interference from your diluent or matrix. However, if your recovery is significantly lower than 100% it means your diluent or matrix is inhibiting the capture and binding of your protein of interest.
But fear not, there are steps you can take to improve your recovery percentage:
- Change the standard diluent: Try using a standard diluent that is similar to your sample matrix. For example, if your samples are culture supernatants following stimulation, try using fresh culture medium. Alternatively, you can use supernatants of unstimulated cells.
- Change the dilution of the sample: If the level of the protein will be sufficient for detection, dilute samples to reduce the interfering component in the sample. For example, if you started with neat samples, try diluting 2-fold in standard diluent or sample matrix.
For more information about spike and recovery experiments visit Spike and Recovery Assessment for ELISA. Here you will find step-by-step instructions to perform a spike and recovery experiment and other protocols to consider when developing your own ELISA.
If you are interested in a ready-to-use ELISA kit please visit our ELISA Selection Tool. We offer a broad menu of over 1,000 ready-to-use ELISA kits that provide accurate, consistent results.
Editorial: David’s Ph.D. and postdoctoral work focused on the study of G protein-coupled receptor pharmacology and thrombosis. Following his academic training, he led Luminex-based multiplexed immunoassay platform development efforts at a Luminex partnering company. In 2009, David joined Thermo Fisher Scientific (formerly Life Technologies) as a senior scientist working on the development of novel immunoassay platforms (e.g., ProtoPlex Immune Response Assay). Working on next-gen immunoassay technologies, David is interested in working with translational investigators and key opinion leaders to identify serum-based biomarkers in cancer, autoimmunity, and inflammation. Beyond his R&D responsibilities, David is dedicated to platform strategy and the Thermo Fisher Scientific antibody content roadmap. https://www.thermofisher.com/us/en/home/support/s2s/david-bourdon.html
Jamie Boden, Technical Writer, IMS: Science has been her passion since childhood; she knew at an early age that she wanted to pursue a career in science where she could learn about how humans were connected, down to the cellular level. Jamie is a biologist turned technical writer with experience ranging from the development of handbooks to webpages and everywhere in between. Jamie strongly believes that science should be available to everyone, because it affects every person, animal and plant on earth.
Stay tuned for more articles from Jamie in the near future!