Modernization of Monoclonal Antibody Screening and Protein-interaction Assays

Citation

Anaya, Michael Aurelio (2021) Modernization of Monoclonal Antibody Screening and Protein-interaction Assays. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/xy05-bb64. https://resolver.caltech.edu/CaltechTHESIS:06072021-233800139

Abstract

In this research, multiplexed bead-based technology was employed to develop a high-throughput monoclonal antibody production method and to refine a protein-protein interaction (PPI) assay for interactome screening. Hybridoma supernatants produced from mice injected with multiple antigens were screened with color-coded, antigen-coupled beads in a semi-automated workflow. Two monoclonal antibodies, each demonstrating high specificity and strong binding, were produced. To our knowledge, these results are the first demonstrated usage of multiplexed suspension bead-based screening as a critical component of high-throughput antibody production. The technology was also utilized as a PPI assay due to its numerous advantages over ELISA-based screens. We studied interactions of the extracellular domains of the Beat and Side protein families, whose members control neuromuscular specificity in Drosophila melanogaster and form a highly-connected interaction network. We demonstrated that a screen utilizing avidin-captured bait was superior to a screen utilizing Protein A-captured bait and deorphanized five proteins within the network, namely Beat 1b, Beat 3a, Beat 3c, Side 5, and Side 8.

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