By: Parmeet Dhillon Date: May 2nd 2025

Literature Review Findings

There is insufficient mention of quantitative methods for assessing accuracy for $k_{a}$ or $k_{d}$ estimates using BLI/SPR
Qualified Biolayer Interferometry Avidity Measurements Distinguish the Heterogeneity of Antibody Interactions with Plasmodium falciparum Circumsporozoite Protein Antigens
- This paper suggests that accuracy cannot be assessed due to the absence of pre-existing BLI data for a specific analyte and ligand pair, similar to the notion that Tong communicated in abc-of-kd-accuracy.pdf
- Beyond this mention, no other steps were taken to assess the accuracy of the novel BLI assay that was being proposed

Tong’s work on $K_{D}$ suggests that, accuracy can be quantitatively assessed, even if the true $K_{D}$ value is unknown.

To investigate this:

I will follow a similar logical workflow that was previously used to propose $A C I - K_{D}$
This will involve the following
- recognizing the presence of systematic errors in common BLI methods
- numerically relating the systematic errors of variables to the systematic errors of $k_{a}$ and $k_{d}$ , using the common method detailed in Common Method for Kinetic Determination

Potential Sources of Systematic Error

FURTHER CONFIRMATION IS REQUIRED:

R_{t} = R_{a} \times e^{- k_{d} t} + R_{t \to i n f}

There are no variables which are being substituted in the equation, $R_{a}$ and $k_{d}$ are both fitted for, via nonlinear regression

R_{t} = {\frac{C k _{a} R _{ma x} ( 1 - e ^{- ((C k_{a} + k_{d}) t)}}{C k _{a} + k _{d}}} + R_{i}

systematic error of analyte concentration, C
systematic error of estimated $k_{d}$ , $k_{d - d e t}$
For $R_{ma x}$
- Although $R_{ma x}$ is generally fitted via nonlinear regression, a theoretical value may be used as a constant for constrained fitting of $k_{a}$
- In the cases where a theoretical value of $R_{ma x}$ is determined using the molecular weights of the analyte and ligand, systematic errors may arise

Mass Transfer: If diffusion to the interaction surface is slower than the rate of binding to the ligand, the diffusion rate becomes a bottle neck
- Mass transport lowers the estimated k_a value
- Solving this includes reducing the surface density of ligand (ensures more local ligand is available for continual binding), or in SPR, increasing analyte injection flow rate (improves convection)
- Source
High ligand density on sensor: rebinding of analyte to ligand on sensor may cause an underestimated k_d value