Wavelength-Dependent Fluorescent Immunosensors via Incorporation of Polarity Indicators near the Binding Interface of Antibody Fragments.
Anal Chem
; 91(12): 7631-7638, 2019 06 18.
Article
em En
| MEDLINE
| ID: mdl-31117403
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos de Imunoglobulinas
/
Técnicas Biossensoriais
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
Austrália