Try before you buy: a new method for validating antigen probes used in B and T cell research

From the Taylor Lab, Vaccine and Infectious Disease Division

With the ever-increasing pace of biological research, there is a plethora of headlines describing novel and fascinating discoveries about our natural world. What’s easy to overlook, however, is the large body of methodological research devoted to developing and validating the technologies used to make those discoveries—to create and sharpen the tools without which these discoveries could not be made. This crucial work is exemplified in a recent publication from the Taylor Lab in the Fred Hutch Vaccine and Infectious Disease Division, which describes the development of a method to test the reliability of a common reagent utilized in the B cell immunology field.

The Taylor Lab studies B cells—the immune cells which primarily function by recognizing portions of a pathogen (called antigens) and producing specific antibodies against these antigens. The specificity of a B cell towards an antigen is mediated by its B cell receptor (BCR), which binds the antigen and stimulates the B cell to proliferate and differentiate into antibody-producing plasma or memory B cells. To study B-cells specific to an antigen of interest, scientists need a tool to detect and isolate them from tissue samples. One common method for accomplishing this is to create a probe consisting of the antigen of interest conjugated to a fluorophore (or to an intermediary molecule which can then be bound to a fluorophore). These probes exploit the BCR-antigen interaction to identify antigen-specific B cells: in a tissue sample incubated with the probe, those B cells which recognize the antigen of interest will bind the probe and be ‘tagged’ with the fluorophore. These B cells can then be detected using a technique like flow cytometry to quantify and even isolate them from the rest of the tissue for further study. While this method makes it possible to study sometimes rare populations of antigen-specific B cells, it also poses a notable challenge: probes are often custom-made by researchers using disparate methods, and it remains difficult and time-consuming to validate their effectiveness before using them on precious tissue samples. All of this means that probe quality varies widely between different laboratories, and that two laboratories using different probes to detect the B cells from the same tissue sample may end up with different results!

In their study published in the Journal of Immunology, a research team led by M3D graduate student Kristin Fitzpatrick of the Taylor Lab set out to address this methodological concern. First, they investigated the effect of storage method on probe performance. Using a consistent protocol, they created batches of probes against the receptor binding domain (RBD) of SARS-CoV-2, stored probe samples at several temperatures for varying lengths of time and tested the probes’ effectiveness at labelling B cells from pooled mouse lymph node and spleen tissue using flow cytometry. This analysis revealed a sharp decline in binding for probes stored in the fridge (4°C) compared to those stored in the freezer (-20°C), but it also revealed considerable batch-to-batch performance variability, even among probes stored at -20°C.

A schematic depicting the experimental setup for using multiple fluorophores to multiplex antigen probe validation (described in the article)
A schematic illustrating the use of beads conjugated to different fluorophores to multiplex antigen probe quality assessment. Image taken from the publication and created using Biorender.

This result suggested that probe storage at -20°C wasn’t enough to ensure reliable performance—researchers needed a convenient tool to assess probe quality before an experiment; ideally, one which didn’t rely on precious (and biologically variable) tissue samples. Towards this aim, Dr. Fitzpatrick and colleagues leveraged a synthetic bead technology already employed in flow cytometry and adapted it for antigen probe validation. Instead of using tissue containing antigen-specific B cells, their method uses synthetic beads conjugated to antibodies against the antigen of interest: by incubating a sample of antigen probe with these antibody-conjugated beads and measuring subsequent binding using flow cytometry (with appropriate controls), the team is able to measure the performance of the probe—before, after, or even during an experiment using the probe on actual tissue samples. Since these synthetic beads can be produced en masse, this method is a first step towards a standardized quality control for antigen probes.

Importantly, the antigen-antibody relationship is not one-to-one; one antigen can be bound by multiple distinct antibodies, so ideally, a robust antigen probe validation assay would be able to efficiently measure the probe’s performance against a mixture of antibodies. Luckily, the team’s bead-based assay is easily able to accommodates this: by including a mix of beads conjugated to different antibodies and corresponding fluorophores, Dr. Fitzpatrick and colleagues measured the performance of the RBD probe against a panel of eight RBD-specific antibodies, which revealed previously hidden variation in probe performance and furthered the utility of this validation method. Gratifyingly, the performance (assessed using beads) of the various batches of probe matched their performance on tissue samples!

The team applied their new validation method to their RBD probe to reach several interesting conclusions, including 1) that probe performance varied significantly even when different antibodies were used against the same antigen, and 2) that the poor probe performance when stored at 4°C likely has more to do with the stability of the fluorophore than the antigen itself. Importantly, their method is applicable to more than just B cells: when a preprint of this work was first posted to Twitter, Dr. Pam Rosato from Dartmouth College quickly reached out and her group collaborated with the Taylor Lab to adapt this approach for use with the type of probe utilized to identify antigen-specific T cells. Overall, they present a robust and efficient method for benchmarking antigen probe quality which—if adopted—can bring valuable standardization to the B cell immunology field, enable easier collaboration among research groups, permit cross-validation of results across different studies, and facilitate continued, reproducible discoveries.

The spotlighted research was funded by the National Institutes of Health, a Fast Grants award, and Fred Hutchinson Cancer Center COVID Pilot Award.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Justin Taylor contributed to this study.

Fitzpatrick, K. S., Degefu, H. N., Poljakov, K., Bibby, M. G., Remington, A. J., Searles, T. G., Gray, M. D., Boonyaratanakornkit, J., Rosato, P. C., & Taylor, J. J. (2023). Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes. The Journal of Immunology, 210(8), 1156–1165.