Capture-stabilize approach for membrane protein SPR assays

Area of Science:

• Biochemistry
• Immunology
• Biotechnology

Background:

• Surface plasmon resonance (SPR) is crucial for antibody-antigen interaction analysis.
• Studying membrane proteins, like G protein-coupled receptors (GPCRs), presents challenges due to their complex structure and native conformation requirements.
• Existing methods for membrane protein analysis via SPR are often limited.

Purpose of the Study:

• To develop a robust method for measuring antibody binding kinetics to intact membrane proteins using SPR.
• To overcome the challenges of immobilizing membrane proteins while maintaining their native structure.
• To provide a reliable platform for analyzing antibodies against membrane proteins and related structures.

Main Methods:

• Purification and affinity capture of His-tagged CXCR5 (a GPCR) onto a Biacore chip.
• Stabilization of the captured receptor using limited cross-linking to preserve native conformation and ligand binding.
• Application of standard Biacore kinetics assay methodology with low pH regeneration.
• Extension of the capture-stabilize approach to virus-like particles for analyzing antibodies against CD52 (a GPI-anchored protein).

Main Results:

• The developed method successfully immobilizes intact membrane proteins, retaining ligand binding activity.
• Accurate antibody kinetics assays were performed using the stabilized receptor.
• The whole receptor assay demonstrated advantages over peptide-based binding assays.
• The approach was validated for virus-like particles, analyzing antibodies against CD52 in a native membrane environment.

Conclusions:

• Chemically stabilized chip surfaces offer a groundbreaking solution for membrane protein SPR assays.
• This method enables reliable kinetic analysis of antibodies against intact membrane proteins and viral particles.
• The technique significantly advances the study of membrane protein-antibody interactions in their native context.