We have published several detailed research protocols on our website to help you improve cell performance and optimize your experimental outputs. 

Two of our protocols have garnered questions from our customers and readers. Namely, what’s the difference between the Recall Antigen Assay and the Antigen-Specific T Cell Assay

In this post, we explain the different uses cases for each assay and provide additional research tips to help you get started.


A Tale of Two Assays

At first glance, these two assays seem similar, but take a closer look to see the significant differences and unique considerations for each. 

 Recall Antigen AssayAntigen-Specific T Cell Assay
PurposeTests PBMC for reactivity to recall antigensMeasures the effects of drugs and antibodies on the immune response
Cells RequiredPrimary PBMC, not cultured cellsCultured T cell lines
Other MaterialsTetanus toxoid, CMV antigen, culture medium, ELISA kit Antigen-presenting cells, peptide antigen for the ASTC, culture medium, mitomycin C
Incubation Time4 days1–4 days
VariablesDonor health, diet, time of collectionResponse may vary by T cell line
ProsCloser to in vivo behavior Easier to monitor T cell of interest, greater signal:noise ratio

Tips for Running a Recall Antigen Assay

The key to running a successful recall antigen assay is to use primary PBMC that are kept frozen until you’re ready to start your assay. PBMC have a short lifespan, so thawing the cells too early or maintaining them in culture can reduce their viability. Because you will be thawing and using primary PBMC immediately and not after culture, the experimental conditions will be closer to in vivo conditions. 

Another consideration is the antigen to be used. We routinely use tetanus toxoid and CMV antigens; we do not use short peptides. The response to individual epitopes is usually very low but using a full-length protein will activate enough T cells to measure responses. This also eliminates the need to match a peptide to the HLA type of the donor PBMC.

Something to keep in mind is that the recall antigen assay measures the function of the cells at the time of sample collection from the donor. Cell function can vary — even when cells are collected from the same donor — depending on their health condition, diet, and other factors at the time of collection. Be sure to purchase enough vials of a single lot to be able to repeat your experiment. 

More Tips From Our Lab

  • Culture Timing: Our protocol suggests a 4-day culture, which is not necessarily when you will see peak cytokine concentration. Optimal timing depends on your antigen of interest and the cytokine being measured. 
  • Culture Medium: Our protocol lists X-VIVO 15®, but you can use other media such as RPMI 1640 supplemented with 5% fetal calf serum. For more culture media considerations, read this case study.  
  • ELISA Kits: Several kits are available to measure both TNFα or IFNγ. 
  • Cytokine Production: The rate of cytokine production varies based on antigen and time.


For more in-depth information on the recall antigen assay, including how we concluded that the assay is consistent over time, read this case study. 


Tips for Running an Antigen-Specific T Cell Assay

The antigen-specific T cell assay measures T cell activation in vitro using cultured T cell lines, which ensures a quick response to the antigen stimulus and not any other antigens. 

The primary consideration when gathering materials to run this assay is that the antigen-specific T cell line you use must be specific to your antigen of choice. This matching makes it easy to monitor only the T cells of interest and gives your results a greater signal:noise ratio.

Note that different T cell lines may have varying responses to the same antigen and that your outcomes only reflect the function of the cell population used in your experiment. Test multiple cell lines to account for this variability and be able to generalize the results.  

More Tips From Our Lab

  • Antigen-Presenting Cells: You can use B-LCL or PBMC as antigen-presenting cells, but those require the addition of mitomycin C, a hazardous material. Alternatively, you can use irradiated PBMC, which does not require mitomycin C handling. 
  • Cytokine Production: If only measuring cytokine production, you do not need to inactivate the antigen-presenting cells first. Collect culture medium for testing after 1–2 days.
  • Proliferation: Day 4 is the best time to measure CD4+ and CD8+ T cell proliferation. Note that to measure CD8+ T cell proliferation, you will need to add low amounts of cytokines (IL-2, IL-12, or IL-15) at the start of culture. 
  • Special Note: You can measure both cytokine production and proliferation from the same assay.

See how we tested for cytokine production in this drug candidate. 


Ready to Run?

With the right materials and background knowledge, you are ready to run your own recall antigen assay or antigen-specific T cell assay. If you encounter any roadblocks or would like us to run your experiments for you, ask our research team for help.

If you need any of the immune cells or reagents mentioned in these protocols, browse our inventory or submit a custom order request.

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