Multiple epitope presentation and surface density control enabled by chemoselective immobilization lead to enhanced performance in IgE-binding fingerprinting on peptide microarrays

Alessandro Gori, Marina Cretich, Renzo Vanna, Laura Sola, Paola Gagni, Giulia Innocenti-Bruni, Marta Liprino, Furio Gramatica, Samuele Burastero, Marcella Chiari

Research output: Contribution to journalArticle

Abstract

Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (Kd).

Original languageEnglish
JournalAnalytica Chimica Acta
DOIs
Publication statusE-pub ahead of print - Jun 2017

    Fingerprint

Keywords

  • Click chemistry
  • IgE detection
  • Multiple epitope ligands
  • Multivalency
  • Peptide microarrays
  • Polymer coating
  • Surface plasmon resonance imaging

ASJC Scopus subject areas

  • Analytical Chemistry
  • Environmental Chemistry
  • Biochemistry
  • Spectroscopy

Cite this