As a biotech researcher for many years, one of lab techniques I used quite often was the Enzyme-Linked Immunosorbent Assay (ELISA for short). For many years, my research focused on understanding the activities of molecules of the immune system called interleukins (part of the cytokine family of molecules). Some of my work included focusing on understanding the biology of a interleukin called interleukin-17A (IL-17A). I maintained a line of mouse fibroblast cells (NIH-3T3 cells) which had been shown would respond to IL-17A and produce IL-6 (another interleukin), measurable by ELISA (click here for the ELISA procedure). If we stimulated the cells with IL-17A along with small amounts of other molecules (TNFα or IL-1β), we would see absolutely massive amounts of IL-6 released – well beyond what would be produced by the cells in response to any one of those molecules alone. At the time, we used this concept to screen blocking antibodies against the IL-17A receptor (IL-17RA) which were then used in a variety of mouse models of different diseases. Fast forward to today: scientists are accumulating data that the cytokine storm observed in some of the sickest COVID-19 patients may actually the result of those patients releasing too much IL-6, perhaps as a result of the activity of IL-17A released by the body as part of the defense against the virus. In fact, there is a clinical trial underway for an antibody to IL-6 called tocilizumab.
With all that as background, it’s time to focus our learning around the ELISA. For starters, if you are using an ELISA kit (like the IL-6 kit I linked to above), most of your assay reagents come packaged up in a tidy little box, and most of the reagents arrive as solids. To use the reagents, you need to add specific volumes of specific solutions (solution concentration!). After preparing the ELISA plates to receive samples, the protein standard must be diluted to the proper starting concentration. Then the protein standard is serially diluted to generate a standard curve. The samples are also often serially diluted. When the assay is complete, if all goes according to plan, you can use the standard curve to determine the concentration of protein in your samples.
Your turn! While not required, you are highly encouraged to work through the HHMI Biointeractive Immunology Virtual Lab. To guide your learning, complete the Immunology Virtual Lab Worksheet and earn +30 bonus points in the lab report category of your semester grade.
Return to Week 36 – Solution Concentration and continue working.