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Functional V(D)J Analysis from Antigen-Specific B cells

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Research Summary:  Antibodies are among the most widely used biological reagents in research and medicine. This contributes to $700 million dollars in annual spending in the United States alone. Reproducibility of these reagents and the results they achieve however remains poor [1]. Recent studiesshow the need to validate monoclonal antibodies as post-translational modifications of certain proteins can affect the binding ability of these widely used reagents [2]. Furthermore, much of these products are nonfunctional or bind off-targeted antigens, resulting in the loss of nearly half of all money spent on these ineffective reagents. This has raised concerns and it has been found that many preclinical therapeutic results are not reproducible and need to be validated [3]. For these reasons, many scientists use only the most widely cited hybridoma clones and increasingly are calling for the sequencing validation of the antibodies they use and production of recombinant antibodies (rAb). Therefore, a paradigm shift towards rAb technology may help to elevate the complications associated with the development of prophylactic and therapeutic strategies for infectious diseases.  We recognize and agree with this movement as a way to increase reproducibility, reduce expenditure and replace the use of animals for hybridoma production. Additionally, expressing these proteins recombinantly will provide unlimited quantities of antibodies while eliminating the difficulties inherent to the maintenance of genetically unstable hybridomas.

This proposal builds upon preliminary data to further investigate the humoral and cellular responses against emerging infectious pathogens.  The incidences of Ebola, Zika, and recently the outbreak of COVID-19, the illness caused by SARS Cov-2 virus as well as other cases of emerging and re-emerging infectious diseases highlight the need for more collaborative efforts to tackle these diseases. The objective of this study is to seek out the rare B cells that make the unique antibodies that have the potential to mature into broadly neutralizing antibodies.  Therefore, we propose utilizing high-throughput single cell B cell receptor sequencing after antigen exposure to allow for the identification of an enormous diversity of full length antibody heavy- and light-chain variable regions from individual B cells.  The V(D)J transcriptome profiles from antigen-specific B cells will allow us to gain insights of genotype, clonal frequency, quality and function resulting during vaccination to inform the success or failure of immune protection.  The significance of this project will help increase our understanding of antibody diversity and improve the challenges of diagnosis and treatment production in addition to prophylactic and therapeutic strategies. 


  1. A. Bradbury and A. Plückthun, “Reproducibility: Standardize antibodies used in research,” Nature, vol. 518, no. 7537, pp. 27–29, Feb. 2015, doi: 10.1038/518027a.
  2. K. A. Janes, “Fragile epitopes—Antibody’s guess is as good as yours,” Sci. Signal., vol. 13, no. 616, p. eaaz8130, Jan. 2020, doi: 10.1126/scisignal.aaz8130.
  3. C. G. Begley and L. M. Ellis, “Raise standards for preclinical cancer research,” Nature, vol. 483, no. 7391, pp. 531–533, Mar. 2012, doi: 10.1038/483531a.


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