Stop Sticking with Hybridoma! A Primer on the Three Generations of Antibody Discovery Technology-Antibody Detection Service Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Stop Sticking with Hybridoma! A Primer on the Three Generations of Antibody Discovery Technology

Have you also experienced this dilemma: investing months in painstaking hybridoma development, only to find the antibodies you screened are too immunogenic, or worse, completely unresponsive to that critical conserved target? Before you switch to a different animal model, perhaps it is time to consider a different technological approach.

For antibody development, hybridoma is no longer the only option, and may not even be the optimal one. Antibody discovery has progressed through three generations, yet many are still stuck with the first. This article will guide you through the evolutionary path from "in vivo blind selection" to "in vitro precision screening" and then to "intelligent design."

I. The Foundational Era: Hybridoma Technology

Kohler and Milstein invented hybridoma technology in 1975, enabling the large-scale production of homogeneous monoclonal antibodies, for which they were awarded the Nobel Prize in 1984.

Core Principle: Mice are immunized multiple times with a specific antigen. Their splenic B cells (which produce antibodies) are fused in vitro with myeloma cells (which can proliferate indefinitely). Through limiting dilution, hybridoma cells producing a single antibody are obtained, followed by affinity purification to yield monoclonal antibodies. This technology is classic, with a relatively standardized workflow, and produces intact IgG antibodies suitable for downstream applications.

However, this technology has unavoidable limitations. For example, the final product is a murine antibody, which can trigger strong immune responses when directly used in humans, leading to reduced efficacy and even safety concerns. Because of immune tolerance in mice, it is difficult to generate high-titer antibodies against highly conserved targets. The screening throughput is low and the cycle is long. Humanization is complex and may compromise affinity.

II. The First Revolution: Display Technologies

In the 1990s, the first in vitro display technology—phage display—emerged, shifting the main battlefield of antibody screening from in vivo to in vitro and opening the door to fully human antibodies.

This technology involves inserting exogenous genes into phage coat protein genes. As phages assemble, the proteins are displayed on their surface. The phage library is then incubated with the target antigen to screen for binding phages, establishing a link between genotype and phenotype. Compared to hybridoma technology, phage display bypasses immunization, allowing direct use of human antibody libraries to obtain fully human sequences. It can be applied to any target, including toxins and antigens that are difficult to immunize. Through methods like error-prone PCR, it can mimic somatic hypermutation in vitro to achieve affinity maturation.

This method also has limitations. The screening process occurs in vitro, and the resulting antibodies may be less stable or developable than those naturally selected in vivo. Traditional display technologies often screen heavy or light chains separately before combining them, potentially disrupting the optimal natural pairing found in vivo.

III. The Second Revolution: Single B Cell Technology

Entering the 21st century, with the explosive development of microfluidics, sequencing, and bioinformatics, antibody discovery has entered a precise, high-speed third generation.

This technology directly isolates individual antigen-specific B cells from the peripheral blood of immunized individuals or convalescent patients, and directly obtains the heavy and light chain genes of their antibodies. This approach "captures" from the source, offering the highest fidelity, fully human sequences, and exceptional speed—particularly advantageous in responding to emerging infectious diseases, delivering candidate molecules within days to weeks.

Figure 1^[2]

TekBiotech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform based on phage display and yeast display technologies. We provide high-quality monoclonal antibody development services for scientists worldwide, covering various formats including scFv, VHH, and Fab. Furthermore, we can develop antibodies with diverse functional and structural characteristics, including but not limited to neutralizing antibodies, conformation-specific antibodies, and cross-reactive antibodies. Additionally, TekBiotech offers supporting downstream services such as antibody expression validation, antibody humanization design and validation, antibody affinity maturation, and CAR-T candidate sequence design, meeting the diverse needs of clients for antibody drug development.

References

[1] Raeisi H, Azimirad M, Asadzadeh Aghdaei H, et al. Rapid-format recombinant antibody-based methods for the diagnosis of Clostridioides difficile infection: Recent advances and perspectives. Front Microbiol. 2022;13:1043214.

[2] Schardt JS, Sivaneri NS, Tessier PM. Monoclonal Antibody Generation Using Single B Cell Screening for Treating Infectious Diseases. BioDrugs. 2024;38(4):477-486.

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