An ELISA-Based Screening Platform for Ligand–Receptor Discovery

Sinem Ozgul, Sventja von Daake, Sumie Kakehi, Davide Sereni, Natalia Denissova, Carlie Hanlon, Yuanpeng Janet Huang, John K. Everett, Cuifeng Yin, Gaetano Montelione, Davide Comoletti

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand–receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein–protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand–receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand–receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsA. Joshua Wand
PublisherAcademic Press Inc.
Pages453-475
Number of pages23
ISBN (Print)9780128167625
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Enzymology
Volume615

Fingerprint

Enzyme-Linked Immunosorbent Assay
Synapses
Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry

Keywords

  • Gene synthesis
  • Glycosylation
  • HEK cells
  • Mammalian cell surface proteins
  • NMR spectroscopy
  • Protein–protein interaction screen
  • Small-angle X-ray scattering
  • X-ray crystallography

Cite this

Ozgul, S., von Daake, S., Kakehi, S., Sereni, D., Denissova, N., Hanlon, C., ... Comoletti, D. (2019). An ELISA-Based Screening Platform for Ligand–Receptor Discovery. In A. J. Wand (Ed.), Methods in Enzymology (pp. 453-475). (Methods in Enzymology; Vol. 615). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.10.001
Ozgul, Sinem ; von Daake, Sventja ; Kakehi, Sumie ; Sereni, Davide ; Denissova, Natalia ; Hanlon, Carlie ; Huang, Yuanpeng Janet ; Everett, John K. ; Yin, Cuifeng ; Montelione, Gaetano ; Comoletti, Davide. / An ELISA-Based Screening Platform for Ligand–Receptor Discovery. Methods in Enzymology. editor / A. Joshua Wand. Academic Press Inc., 2019. pp. 453-475 (Methods in Enzymology).
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Ozgul, S, von Daake, S, Kakehi, S, Sereni, D, Denissova, N, Hanlon, C, Huang, YJ, Everett, JK, Yin, C, Montelione, G & Comoletti, D 2019, An ELISA-Based Screening Platform for Ligand–Receptor Discovery. in AJ Wand (ed.), Methods in Enzymology. Methods in Enzymology, vol. 615, Academic Press Inc., pp. 453-475. https://doi.org/10.1016/bs.mie.2018.10.001

An ELISA-Based Screening Platform for Ligand–Receptor Discovery. / Ozgul, Sinem; von Daake, Sventja; Kakehi, Sumie; Sereni, Davide; Denissova, Natalia; Hanlon, Carlie; Huang, Yuanpeng Janet; Everett, John K.; Yin, Cuifeng; Montelione, Gaetano; Comoletti, Davide.

Methods in Enzymology. ed. / A. Joshua Wand. Academic Press Inc., 2019. p. 453-475 (Methods in Enzymology; Vol. 615).

Research output: Chapter in Book/Report/Conference proceedingChapter

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AU - Kakehi, Sumie

AU - Sereni, Davide

AU - Denissova, Natalia

AU - Hanlon, Carlie

AU - Huang, Yuanpeng Janet

AU - Everett, John K.

AU - Yin, Cuifeng

AU - Montelione, Gaetano

AU - Comoletti, Davide

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N2 - Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand–receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein–protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand–receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand–receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.

AB - Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand–receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein–protein interaction (PPI) complexes. Here, we provide a detailed protocol for a 384-well ELISA-based PPI screening protocol for the identification of novel cell surface ligand–receptor interactions, together with considerations for validation of PPIs by biophysical methods. This PPI screen has been developed and tested for discovery of novel ligand–receptor pairs between human synaptic adhesion proteins, believed to play crucial roles in many steps of neurodevelopment, from neuronal maturation, to axon guidance, synapse connectivity, and pruning.

KW - Gene synthesis

KW - Glycosylation

KW - HEK cells

KW - Mammalian cell surface proteins

KW - NMR spectroscopy

KW - Protein–protein interaction screen

KW - Small-angle X-ray scattering

KW - X-ray crystallography

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SN - 9780128167625

T3 - Methods in Enzymology

SP - 453

EP - 475

BT - Methods in Enzymology

A2 - Wand, A. Joshua

PB - Academic Press Inc.

ER -

Ozgul S, von Daake S, Kakehi S, Sereni D, Denissova N, Hanlon C et al. An ELISA-Based Screening Platform for Ligand–Receptor Discovery. In Wand AJ, editor, Methods in Enzymology. Academic Press Inc. 2019. p. 453-475. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2018.10.001