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Modernization of Monoclonal Antibody Screening and Protein-interaction Assays


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.


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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Interactome, hybridoma, antibody screening
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biology
Awards:Lucy Guernsey Service Award, 2018
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Zinn, Kai George
Thesis Committee:
  • Wold, Barbara J. (chair)
  • Zinn, Kai George
  • Rothenberg, Ellen V.
  • Bjorkman, Pamela J.
Defense Date:2 March 2021
Record Number:CaltechTHESIS:06072021-233800139
Persistent URL:
Anaya, Michael Aurelio0000-0002-6944-3614
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14259
Deposited By: Michael Anaya
Deposited On:06 Mar 2023 19:16
Last Modified:08 Nov 2023 17:58

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