Kinetic titration series with biolayer interferometry

Abstract

Biolayer interferometry is a method to analyze protein interactions in real-time. In this study, we illustrate the usefulness to quantitatively analyze high affinity protein ligand interactions employing a kinetic titration series for characterizing the interactions between two pairs of interaction patterns, in particular immunoglobulin G and protein G B1 as well as scFv IC16 and amyloid beta (1-42). Kinetic titration series are commonly used in surface plasmon resonance and involve sequential injections of analyte over a desired concentration range on a single ligand coated sensor chip without waiting for complete dissociation between the injections. We show that applying this method to biolayer interferometry is straightforward and i) circumvents problems in data evaluation caused by unavoidable sensor differences, ii) saves resources and iii) increases throughput if screening a multitude of different analyte/ligand combinations.

Figure 1. Evaluation of (A) kinetic titration series and (B) parallel sensor kinetics with rabbit IgG binding to GB1 in BLI.

The sensorgrams show the interaction of IgG (analyte) with GB1 (ligand). Applied analyte concentrations were: 0.5, 0.25, 0.125, 0.0625 and 0.03125 µM. The fits are indicated by the red lines, whereas the sensorgrams are shown in black (A) and blue (B). The residuals of the fits are plotted below the respective sensorgram. All other experiments are shown in File S1.

Figure 2. Comaprison of kinetic titration series (A–C) and parallel sensor kinetics (D–F) with scFv IC16 binding to Aβ(1–42) in BLI.

The sensorgrams show the interaction of scFv IC16 (analyte) with C-terminally biotinylated Aß(1–42) (ligand). The amount of ligand was increased from 0.13 nm (A, D), 0.41 nm (B, E) and 1.01 nm (C, F). Applied analyte concentrations were: 2.4, 1.2, 0.6, 0.3 and 0.15 µM. The fits are indicated by the red lines, whereas the sensorgrams are shown in blue. Each kinetic titration series was reproduced five times. The residuals of the fits are plotted below the respective sensorgram.