Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement.
Duy Tien TaRosario VanellaMichael A NashPublished in: ACS applied materials & interfaces (2018)
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.
Keyphrases
- amino acid
- highly efficient
- tissue engineering
- induced apoptosis
- binding protein
- protein protein
- liquid chromatography tandem mass spectrometry
- mass spectrometry
- loop mediated isothermal amplification
- high performance liquid chromatography
- pet imaging
- high resolution
- endoplasmic reticulum stress
- single molecule
- staphylococcus aureus
- liquid chromatography
- gas chromatography
- dna binding
- positron emission tomography
- tandem mass spectrometry