Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement

Area of Science:

• Biomolecular Engineering
• Protein Chemistry
• Bioorthogonal Chemistry

Background:

• Artificial multiprotein complexes are valuable reagents in biomolecular engineering.
• A key limitation is the lack of molecular scaffolds for site-specific multicomponent assembly.
• Existing methods often lack efficiency and orthogonality for complex assembly.

Purpose of the Study:

• To synthesize novel bioorthogonal elastin-like polypeptide (ELP) scaffolds.
• To enable site-specific assembly of multiple single-domain antibodies (SdAbs) onto ELP scaffolds.
• To demonstrate the utility of these multi-antibody ELP complexes in biological applications.

Main Methods:

• Synthesized ELP scaffolds with periodic l-azidohomoalanine residues for azide conjugation.
• Utilized strain-promoted alkyne-azide cycloaddition (SPAAC) for site-specific antibody attachment via DBCO-functionalized SdAbs.
• Incorporated Sortase A and ybbR tags for additional orthogonal modifications with small molecules and peptides.

Main Results:

• Successfully synthesized multi-antibody ELP complexes in a one-pot reaction using bioorthogonal chemistry.
• Demonstrated enhanced performance in sandwich immunoassays for mCherry protein, showing 2.3- to 14.3-fold sensitivity improvement and lower detection limits in human plasma.
• Applied dual-labeled ELP complexes for effective cytometric analysis of live eukaryotic cells.

Conclusions:

• Developed a versatile platform for creating artificial multiprotein complexes using bioorthogonal ELP scaffolds.
• The multi-antibody ELP complexes significantly enhance immunoassay sensitivity and enable advanced cellular analysis.
• This approach leverages multivalency effects for improved immunospecific binding interactions.