Biotechnology Journal - 2019 - Zhou - High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing

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

Introduction

  • Purpose: Improve antibody detection and modification through a novel method using a small antibody-binding protein.

  • Background: Antibodies are essential in research and medicine due to their specificity and high affinity for antigens.

  • Challenges: Direct chemical labeling of antibodies results in poorly defined modifications. Traditional antibody-binding proteins (bABPs) are complex and inefficient.

Innovative Solution

  • Adapter Protein: Development of a bivalent antibody-binding protein by joining two Z domains with a flexible linker.

    • Key Feature: Linker enhances affinity via simultaneous binding.

  • Circularization: The protein is circularized with a split intein, termed "lasso."

    • Binding Affinity: Lasso binds human IgG1 (KD = 0.53 nM), significantly improving the dissociation rate compared to existing methods.

Antibody-Binding Proteins (ABPs)

  • Types of ABPs: Commonly sourced from

    • Staphylococcus aureus (SpA)

    • Group C and G streptococcal proteins (protein G)

    • Peptostreptococcus magnus (protein L)

  • Historical Context: Development and engineering of ABPs have progressed, enabling applications like affinity purification and enzyme-linked immunosorbent assay (ELISA).

Mechanism and Design

  • Z Domain Development: The Z domain is a single-domain variant of SpA with favorable properties such as resistance to aggregation.

    • Weakness: Existing ABDs exhibit low affinity and short-lived interactions.

  • Lasso Design: The lasso shows 10–12 fold higher affinity than single Z domains and allows for effective site-directed modifications.

Experimental Section

  • Plasmid and Vector Construction: Various vectors engineered to express and purify the lasso construct.

  • Expression and Purification: Utilization of E. coli strains for efficient protein expression.

  • Biotinylation: Achieved via AviTag with BirA coexpressed.

    • Verification: Successful biotinylation confirmed through gel electrophoresis.

Functional Characterization

  • Fluorescence Labeling: Incorporation of fluorescein-5-maleimide to enable tracking.

  • Yeast Display: Demonstrated effective binding and affinity measurements using flow cytometry.

Results

  • Affinity Measurements: Lasso showed superior performance in comparison to Z and ZZ.

    • KD Values: Z (9.6 nM), ZZ (5.5 nM), Lasso (0.53 nM).

  • ELISA Performance: The lasso resulted in stronger reporter signals than secondary antibodies.

    • Limit of Detection (LOD): 0.13 ng/mL for the lasso vs. 1.6 ng/mL for secondary antibody methods.

Applications and Future Directions

  • Protease-Sensitive Lasso: Incorporation of protease recognition sequences to develop localized sensors for protease activity.

  • Fluorescence Microscopy: Lasso used to detect cell-bound antibodies with specificity.

  • Conclusion: The lasso represents a novel tool for antibody modification and detection without compromising function, opening avenues in biosensing applications.