When Hybridomas Die: Recovering Critical Reagents with Antibody Sequencing

Natalia G. Sampaio, Linden J. Gearing, Antonio G. Dias Junior et al. MDA5 guards against infection by surveying cellular RNA homeostasis (2025), PREPRINT (Version 1) available at Research Square. https://doi.org/10.21203/rs.3.rs-6466919/v1

Key Takeaways

• Researchers required a unique monoclonal antibody to study MDA5’s role in viral RNA sensing, but the original hybridoma cell line was lost.
• Using Rapid Novor’s REmAb® sequencing service, the full sequence of the antibody was rescued by de novo protein sequencing using mass spectrometry and reproduced recombinantly.
• This enabled critical experiments that uncovered MDA5’s function in monitoring RNA processing fidelity and detecting infection via splicing perturbations.

Summary

Melanoma differentiation–associated protein 5 (MDA5) is an innate immune RNA sensor that detects viral and pathogen-derived RNAs, but the precise nature of its RNA agonists has remained unclear.

In 2025, Linden J. Gearing, Antonio G. Dias Junior, and colleagues from the Hudson Institute of Medical Research and the University of Oxford set out to map RNAs bound by MDA5 during viral infection using single-nucleotide resolution crosslinking and immunoprecipitation (iCLIP).

To do so, the team raised monoclonal antibodies against MDA5 and identified two clones: antibody A (clone 16), effective against native MDA5, and antibody B (clone 22), targeting denatured MDA5; both critical reagents for their iCLIP workflow.

When the hybridoma cells producing antibody B were non-recoverable, the team faced the loss of this essential reagent. To overcome this challenge, they employed Rapid Novor’s REmAb® monoclonal antibody sequencing service. Through de novo sequencing by protein mass spectrometry, Rapid Novor rescued the full amino acid sequence of antibody B. The sequence was then used to recombinantly express the antibody in mammalian cells, ensuring it could be securely and indefinitely reproduced.

This reagent rescue allowed the researchers to complete their iCLIP experiments, ultimately revealing that MDA5 monitors RNA processing fidelity and detects infection through perturbations in post-transcriptional events such as splicing, establishing a new paradigm of innate immune “guarding” by RNA sensors.