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A better way to select promising antibodies

Scientists develop highly specific monoclonal antibodies by screening hundreds of candidates to identify those that strongly and precisely bind target antigens. Traditionally, scientists used biochemical assays such as enzyme-linked immunosorbent assay for antibody screening, but flow cytometry has made this once lengthy and tedious process easier and more efficient (1). Fluorescence-activated cell sorting (FACS), an advanced form of flow cytometry, is a powerful technology that helps researchers sift through diverse cell populations to isolate cells producing the most potent antibodies (2). Today, FACS plays a crucial role in therapeutic antibody development, with more than 100 monoclonal antibodies approved for human therapies, plus at least 140 more in late-stage development (3,4).

Merging cells for unlimited antibody production

The antibody generation process starts with hybridomas, cells generated by fusing immortalized myeloma cells with antibody-producing B cells derived from animals immunized against the antigen of interest or from humans who recovered from illness (2). Stable hybridoma cells produce unlimited amounts of membrane-bound and soluble antibodies that recognize the same single antigen (5).

Identifying the strongest antibodies using fluorescent tags

Scientists label the target antigen in solution with fluorescent tags and introduce it to cultured hybridoma cells to find the hybridoma cells with antibodies that strongly and specifically bind the target antigen (6).

Sorting cells by fluorescence strength

Scientists then analyze the fluorescent signals of the hybridoma cells as they pass through a laser beam during FACS. Cells expressing antibodies that bind the fluorescent antigen fluoresce with greater intensity than cells with weak or no antigen binding (6).

A continuous source of soluble antibodies

Scientists can then harvest the culture medium, extract the soluble antibodies, purify them, and validate them for therapeutic purposes, including disease diagnosis or immunotherapy (6,7,8).

Expanding cells expressing the right antibodies

Next, scientists collect and expand the cells expressing antibodies with the highest binding affinity for the antigen. This generates a homogeneous population of cells producing the most potent antibodies.

References

1. Bickerstaff, A.A., et al. A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection. J Virol Methods 142, 50-58 (2007).
2. Robinson, J.P., Ostafe, R., Iyengar, S.N., Rajwa, B., & Fischer, R. Flow cytometry: the next revolution. Cells 12, 1875 (2023).
3. Mullard, A. FDA approves 100th monoclonal antibody product. Nat Rev Drug Discov 20, 491–495 (2021).
4. Kaplon, H., Crescioli, S., Chenoweth, A., Visweswaraiah, J., & Reichert, J. M. Antibodies to watch in 2023. mAbs 15, 2153410 (2023).
5. Sakaguchi, A., et al. Rapid, simple, and effective strategy to produce monoclonal antibodies targeting protein structures using hybridoma technology. J Biol Eng 17, 24 (2023)
6. Akagi, S., Nakajima, C., Tanaka, Y., & Kurihara, Y. Flow cytometry-based method for rapid and high-throughput screening of hybridoma cells secreting monoclonal antibody. J Biosci Bioeng 125, 464-469 (2017).
7. Fei, C., Nie, L., Zhang, J., & Chen, J. Potential applications of fluorescence-activated cell sorting (FACS) and droplet-based microfluidics in promoting the discovery of specific antibodies for characterizations of fish immune cells. Front Immunol 12, 771231 (2021).
8. Berger, M., Shankar, V., & Vafai, A. Therapeutic applications of monoclonal antibodies. Am J Med Sci 324, 14-30 (2002).