Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments

Anal Chem. 2019 Jun 18;91(12):7631-7638. doi: 10.1021/acs.analchem.9b00445. Epub 2019 Jun 5.

Abstract

Herein, we describe a fluorescent immunosensor designed by incorporating an unnatural amino acid fluorophore into the binding site of an EGFR-specific antibody fragment, resulting in quantifiable EGFR-dependent changes in peak fluorescence emission wavelength. To date, immunosensor design strategies have relied on binding-induced changes in fluorescence intensity that are prone to excitation source fluctuations and sample-dependent noise. In this study, we used a rational design approach to incorporate a polarity indicator (Anap) into specific positions of an anti-EGFR single chain antibody to generate an emission wavelength-dependent immunosensor. We found that when incorporated within the topological neighborhood of the antigen binding interface, the Anap emission wavelength is blue-shifted by EGFR-binding in a titratable manner, up to 20 nm, with nanomolar detection limits. This approach could be applicable to other antibody/antigen combinations for integration into a wide range of assay platforms (including homogeneous, solid-phase assay, or microfluidic assays) for one-step protein quantification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / genetics
  • Amino Acids / metabolism
  • Antibodies / immunology
  • Antigen-Antibody Reactions
  • Biosensing Techniques / methods*
  • ErbB Receptors / genetics
  • ErbB Receptors / immunology
  • Fluorescent Dyes / chemistry
  • Humans
  • Immunoassay
  • Immunoglobulin Fragments / chemistry*
  • Immunoglobulin Fragments / immunology
  • Limit of Detection
  • Polymorphism, Single Nucleotide

Substances

  • Amino Acids
  • Antibodies
  • Fluorescent Dyes
  • Immunoglobulin Fragments
  • ErbB Receptors