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Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement.

  • Ta DT Department of Chemistry , University of Basel , 4058 Basel , Switzerland.
  • Vanella R Department of Chemistry , University of Basel , 4058 Basel , Switzerland.
  • Nash MA Department of Chemistry , University of Basel , 4058 Basel , Switzerland.
  • 2018-08-21
Published in:
  • ACS applied materials & interfaces. - 2018
ELP
SPAAC
multiprotein complex
protein polymer
single-domain antibody
Cycloaddition Reaction
Elastin
Humans
Immunoassay
Limit of Detection
Luminescent Proteins
Peptides
English Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4'-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.
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  • English
Open access status
green
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https://sonar.ch/global/documents/15540
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