Antibody binding to a tethered vesicle assembly using QCM-D
Ankit R Patel1, Kay K Kanazawa, Curtis W Frank
1Department of Chemical Engineering, Stanford University, 381 North-South Mall, Stauffer III, Stanford, California 94305-5025, USA.
Analytical Chemistry
|July 8, 2009
Summary
This study quantifies antibody binding to model membranes using quartz crystal microbalance with dissipation monitoring (QCM-D). Researchers developed a method to accurately measure antibody amounts, revealing insights into membrane-protein interactions.
Area of Science:
- Biomembrane research
- Analytical chemistry
- Surface science
Background:
- Bilayer-tethered vesicle assemblies serve as biomimetic platforms for integral membrane protein analysis.
- Quartz crystal microbalance with dissipation monitoring (QCM-D) offers quantitative, label-free study of membrane surface binding.
- Accurate quantification of antibody binding to viscoelastic systems like tethered vesicles remains challenging.
Purpose of the Study:
- To establish an empirical relationship between QCM-D response and bound antibody amount for tethered vesicle assemblies.
- To validate QCM-D findings against independent measurements using an in situ ELISA assay.
- To investigate the viscoelastic properties of the membrane upon antibody binding.
Main Methods:
- Utilized quartz crystal microbalance with dissipation monitoring (QCM-D) to monitor antibody binding.
- Employed a bilayer-tethered vesicle assembly displaying dinitrophenyl (DNP) hapten groups.
- Used monoclonal antidinitrophenyl (DNP) IgG(1) as the model antibody.
- Performed in situ ELISA assays for independent quantification of bound antibody.
Main Results:
- Established an empirical correlation between QCM-D signals and antibody surface density.
- Achieved a saturation surface mass density of approximately 900 ng/cm(2) for bound antibody.
- Demonstrated that a homogeneous, one-layer QCM-D model predicts bound antibody mass accurately near saturation.
- Indicated that a two-layer model is necessary to describe the kinetic dissipation response, suggesting top-layer stiffening upon antibody binding.
Conclusions:
- Developed a robust method for quantifying antibody binding to tethered vesicle model membranes using QCM-D.
- Provided evidence that antibody binding induces structural changes, specifically stiffening of the membrane's top layer.
- Highlighted the utility of QCM-D in studying the dynamics and mechanics of biomolecular interactions at model membrane surfaces.
