High-Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody-Binding Domains

Area of Science:

• Biotechnology
• Protein Engineering
• Immunology

Background:

• Direct chemical labeling of antibodies results in poorly defined modifications.
• Antibody functionalization can be simplified using small antibody-binding proteins as adapters.
• Stabilizing noncovalent antibody complexes without chemical crosslinking is challenging.

Purpose of the Study:

• To engineer a novel, highly stable adapter protein for site-specific antibody functionalization.
• To improve antibody detection sensitivity and simplify antibody-based assays.
• To create a versatile protein tool for antibody conjugation and immobilization.

Main Methods:

• Engineering a bivalent antibody-binding protein by joining two Z domains with a flexible linker.
• Circularizing the protein using a split intein to create the "lasso" adapter.
• Characterizing the binding affinity (K_D) and dissociation rate of the lasso to human IgG1.
• Evaluating the lasso's performance in enzyme-linked immunosorbent assay (ELISA).

Main Results:

• The engineered lasso protein binds human IgG1 with high affinity (K_D = 0.53 nM) and a significantly slower dissociation rate.
• The lasso adapter allows for site-specific conjugation with reporters, biotin tags, and protease sequences.
• In ELISA, the lasso generated a 12-fold lower limit of detection compared to secondary antibodies.
• The lasso demonstrated a stronger reporter signal than conventional secondary antibodies.

Conclusions:

• The circular lasso protein is a highly effective adapter for antibody functionalization and detection.
• Its high affinity, slow dissociation, and conjugation versatility make it valuable for various antibody-based applications.
• The lasso significantly enhances sensitivity in ELISA, offering a promising tool for antibody detection and immobilization.