{"id":8282,"date":"2016-04-12T10:00:07","date_gmt":"2016-04-12T14:00:07","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=8282"},"modified":"2016-05-26T23:35:06","modified_gmt":"2016-05-26T23:35:06","slug":"developing-your-own-elisa-spike-recovery-experiments","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/developing-your-own-elisa-spike-recovery-experiments\/","title":{"rendered":"Developing your own ELISA: Spike & Recovery Experiments"},"content":{"rendered":"

\"Adding<\/a>Editorial provided by David Bourdon, Ph. D. and Sr. R&D Manager & Immunoassay Strategy Lead\u00a0at Thermo Fisher Scientific.<\/strong><\/em><\/p>\n

Recovery is a fundamental biophysical property in the immunoassay developer community. \u00a0As 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. <\/em><\/strong><\/p>\n

Developing your own ELISA can be quite a task! There are many things to consider when building your own immunoassay \u2013 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<\/a>, 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.<\/p>\n

The best way to ensure you are measuring the true concentration is to run a spike-and-recovery experiment<\/a>. 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 \u201cspike,\u201d 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 \u201crecover\u201d upon measurement. Ideally, your results should be as close to 100% recovery as possible.<\/p>\n

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.<\/p>\n

But fear not, there are steps you can take to improve your recovery percentage:<\/p>\n