High Throughput Solution-Based Measurement of Antibody-Antigen affinity and epitope binding

High Throughput Solution-based measurement of anti-body-antigen affinity and epitope binding.pdf

BLI vs SPR

• Antigen consumption is a common concern for SPR, a problem which is solved by BLI

For accurate $K_D$ measurement:

• lowest practical sensor loading density

  • High density may slow down the rate of diffusion of the analyte to the surface
  • As a result, a slower apparent binding rate is observed, rather than the true kinetic rate = negatively affects the accuracy of $k_a$

• multiple concentrations of analyte to minimize mass transport related effects and rebinding

• Rebinding

  • at high sensor loading densities, analyte that can dissociate quickly may rebind to the immobilized ligand, resulting in an underestimated $k_d$ value

Results

• Upon conducting kinetic measurements, it became clear that as the loading density on the sensor increased, the fitted $k_a$ value also increased
• $k_a$ values further increased when a global fit was conducted using multiple antibody concentrations, and a fixed low loading density
  • Even after conducting the following adjustments for optimization, there was a $K_D$ detection limit of 100 picomolar.

For our purposes: the results and relationship between loading density on the sensor does not have any relevance for improving the accuracy of the method