Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross...

Area of Science:

• Biophysics
• Immunology
• Analytical Chemistry

Background:

• Tumor necrosis factor-alpha (TNFα) is a key cytokine in inflammatory and immune responses.
• Understanding TNFα-antibody interactions is crucial for developing targeted therapies.
• Quantitative kinetic data for TNFα-antibody binding in solution is limited.

Purpose of the Study:

• To quantitatively investigate the binding kinetics of TNFα with its specific antibody (anti-TNFα) using dual-color fluorescence cross-correlation spectroscopy (FCCS).
• To determine the association rate constant (k(on)), dissociation rate constant (k(off)), and dissociation constant (K(d)) of the TNFα-anti-TNFα interaction.
• To validate the biophysical measurements using complementary techniques like dynamic light scattering (DLS).

Main Methods:

• Fluorescent labeling of TNFα with Alexa488 and anti-TNFα with Atto647N.
• Dual-color fluorescence cross-correlation spectroscopy (FCCS) to analyze binding kinetics in solution.
• Analysis of fluorescence correlation spectroscopy (FCS) autocorrelation curves to determine diffusion coefficients and hydrodynamic diameters.
• Comparison of FCCS-derived hydrodynamic sizes with native molecule sizes measured by dynamic light scattering (DLS).

Main Results:

• Diffusion coefficients for Alexa488-TNFα and Atto647N-anti-TNFα were determined as 100.06 ± 4.9 μm(2)s(-1) and 48.96 ± 2.52 μm(2)s(-1), respectively.
• Hydrodynamic diameters were calculated as approximately 4.89 ± 0.24 nm for Alexa488-TNFα and 9.99 ± 0.52 nm for Atto647N-anti-TNFα, consistent with DLS measurements.
• Binding kinetics revealed an association rate constant (k(on)) of (1.13 ± 0.08) × 10(4) M(-1)s(-1), a dissociation rate constant (k(off)) of (1.53 ± 0.19) × 10(-3)s(-1), and a dissociation constant (K(d)) of (1.36 ± 0.10) × 10(-7) M at 25 °C.

Conclusions:

• This study presents the first report on the binding kinetics of TNFα-antibody recognition in a homogeneous phase using FCCS.
• The quantitative kinetic data provides valuable insights into the molecular mechanisms governing TNFα-mediated immune responses.
• FCCS is demonstrated as a powerful technology for controlled experiments to study molecular interactions relevant to immunology and drug development.