Modernization of Monoclonal Antibody Screening and Protein-interaction Assays

Author: Anaya, Michael Aurelio

Year: 2021

Degree: Dissertation (Ph.D.)

Advisor: Zinn, Kai George

Committee Members: Wold, Barbara J.; Zinn, Kai George; Rothenberg, Ellen V.; Bjorkman, Pamela J.

Option: Biology

DOI: 10.7907/xy05-bb64

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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