Using a Recall Antigen Assay as a Tool for Understanding Immunity


While there is no industry-accepted protocol for measuring the functionality of peripheral blood mononuclear cells (PBMC), it’s an important test that should be conducted for quality control.

We needed a reliable, reproducible way to measure the functionality of our cryopreserved PBMC, so we developed a custom assay using recall antigens to understand the in vitro activity of our cells. By testing PBMC for immune response to several different antigens over time, we intended to better understand an acceptable range of variation in recall response from PBMC samples from the same donor over time.


  1. Cryopreserved PBMC were thawed in a 37°C water bath and the concentration adjusted to 2.5 x 106 per mL in X-VIVO™ 15 medium.
  2. Cells were added to a round bottom, 96 well plate at 100 uL per well.
  3. Dilutions of antigens and mitogens were prepared in X-VIVO 15 medium. Tetanus toxoid and Cytomegalovirus (CMV) antigens were diluted to 2 ug/mL. Lipopolysaccharide (LPS) was diluted to 200 ng/mL. Phytohemaglutinin (PHA) was titrated to determine the optimal concentration, which we found to be 1 ug/mL.
  4. Antigens were added to the well plate at 100 uL per well in triplicate. 100 uL of medium was added to three wells as a control.
  5. Samples were incubated at 37°C, 6% CO2 for four days. Note: Day four is not the optimal time to measure cytokine concentrations for all of the antigens and mitogens in this assay. Day four was chosen to maximize the chance of seeing a response to all four antigens. (See Figures 1 and 2)
  6. 150 uL of medium was removed from each well and used for cytokine analysis.
Figure 1. IFNγ concentrations for samples containing tetanus toxoid and CMV were measured on culture day one, two, three, and four. Day four may or may not have been peak concentration.
Kinetics of TNF-alpha Production graph
Figure 2. Peak cytokine concentration for samples containing PHA was observed on culture day three using TNFα. Peak cytokine concentration for samples containing LPS was observed on culture day one using TNFα.


  • Recall Response is Highly Reproducible, But Some Variation is Expected

Repeated testing of a reference lot of PBMC using the recall antigen assay resulted in varying levels of cytokine production, even when the lot of antigen, culture medium, and other conditions were kept constant. (See Figure 3)

Due to this variability, the recall antigen assay should not be used to compare separate runs or experiments. Although recall patterns seem to be stable across experiments, the variation makes it difficult to compare results quantitatively.

However, comparisons of results within the same run or experiment are acceptable. We have used this recall antigen assay to evaluate different lots of antigen with high reproducibility. (See Figure 4)

Vial-to-Vial Reproducibility Graph
Figure 3. The response to tetanus toxoid was measured using the same lot of PBMC and the same lot of antigen on five separate occasions. Each experiment reported an immune response to tetanus toxoid, but IFNγ production varied from less than 100 pg/mL to 700 pg/mL.
Comparison of CMV Antigen Lots Graph
Figure 4. The recall antigen assay was used to measure cytokine production of the PBMC samples using two different lots of CMV at varying concentrations. This graph shows lot-to-lot variability for CMV.
  • Donor Response to Recall Antigens Varies Over Time
Donor 287 Graph
Figure 5. The response to tetanus toxoid and CMV antigens were measured using the PBMC samples taken from the same donor at different times over a 20-month period. Except for a lower response in January 2016, donor 287 maintained a robust response to tetanus toxoid and CMV.

The degree of recall response can vary over time as measured by our recall antigen assay, even for the same donor. Be cautious of using the same donor for multiple experiments, as even samples from the same donor can vary depending on factors such as time of collection. Our samples were all collected at approximately the same hour of the day, but other variables can impact the immune response. More research is needed to understand these variables.

As shown in Figure 5, samples from donor 287 collected at different times over a 20-month period had a positive response to both tetanus toxoid and CMV, but the degree of response varied over time even as all other variables were kept constant.

Our experiments have shown that tetanus immunizations have a lasting impact. Most of our donors have a positive response to tetanus toxoid (defined as 100 pg/mL or more), although the degree of response has a wide range of up to 10,000 pg/mL. This range could be attributed to the recency of a booster vaccination.

Compared to a one-time tetanus immunization, the responses to CMV, a chronic infection, were much less variable for donor 287. Because CMV-positive donors have the virus for life, their T cells are more likely to be exhausted by the constant battle to keep the virus in check. This may explain the relative ease of demonstrating the effect of a PD-1 antibody on the in vitro immune response to CMV, and is also a model for response to cancer antigens, which are chronically present and likely to drive T cells to exhaustion.

  • The Recall Antigen Assay Can Be Manipulated to Increase or Decrease the Immune Response
Donor 349 Graph
Figure 6. The response of donor 349 to CMV antigens was measured when Nivolumab (Opdivo®) was added to the culture. IgG4 was used as a negative control for Nivolumab. The secretion of IFNγ was increased 100% or more by this checkpoint inhibitor.

There is great excitement surrounding the progress of targeted therapies that use the immune system to attack cancer cells. The activity of one such drug, Nivolumab, can be demonstrated in our recall antigen assay by measuring the immune response to CMV antigen.

Nivolumab works by intensifying the immune response to cancer. Figure 6 shows that the cultures in our assay containing Nivolumab had a greater response to CMV antigen than the control cultures.

Similar approaches can also be tested using this assay to monitor the impact of drug candidates on the in vitro immune response. Whether you want to boost the response as in cancer or vaccine design or blunt the immune attacks as in autoimmune disease or allergy drug candidates, this assay can be used as a model.


The assay we developed is a reliable measure of immune responses, but some variation is expected. When using our recall antigen assay to test for functionality and immunity, always use a variety of donors and test multiple times to ensure your results are dependable.

Our recall antigen assay has been useful in demonstrating the functionality of PBMC and has also been used as a model for monitoring the effects of test compounds on immune cell activation.

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