Validation of ligand tetramers for the detection of antigen-specific lymphocytes

The study of antigen-specific lymphocytes has been a key advancement in immunology the past few decades. The development of multimerized probes containing antigens, peptide:MHC complexes, or other ligands was one innovation allowing the direct study antigen-specific lymphocytes by flow cytometry. While these types of studies are now common and performed by thousands of laboratories, quality control and assessment of probe quality is often minimal. In fact, many of these types of probes are made in-house and protocols vary between labs. While peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for antigen multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind antibodies specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and antigen tetramers and find considerable batch-to-batch variability in performance and stability over time. This assay can also reveal common production errors such as miscalculation of antigen concentration. Unexpectedly, probes including the fluorochrome allophycocyanin exhibited more rapid performance decline compared to probes including the fluorochrome R-phycoerythrin when both were stored at 4°C. This performance decline was reduced for most, but not all, batches when antigen tetramers were instead stored at −20°C in 50% glycerol. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit lab-to-lab technical variation, and experimental failure due to probe underperformance.