Application note

Measuring the binding affinity of protein–protein interactions in simple and complex backgrounds

Authors
Sebastian Fiedler1, Maren Butz1, Haris Choudhery1 & Sean Devenish1

Microfluidic Diffusional Sizing was used to determine the dissociation constant (KD) of a protein antibody interaction. This was quantified in both a well defined buffer and a cell culture medium with high precision.

Thus, the Fluidity One-W is able to determine binding affinities in a range of backgrounds — potentially reducing the need for time consuming purification steps.

Introduction

Quantitative analysis of protein–protein interactions usually requires time consuming purification steps. Here we show how the Fluidity One-W can assess the binding affinity of proteins in both well defined biological buffers and complex backgrounds with high precision.

The Fluidity One-W was used to measure the dissociation constant (KD) and hydrodynamic radius (Rh), of Protein A (SpA) bound to immunoglobulin G (IgG) in Phosphate buffer solution with 0.05% Tween 20 (PBS-T) and FreeStyle 293 expression media.

SpA is a 42 kDa virulence factor that is produced by the pathogen Staphylococcus aureus. It is composed of five homologous IgG binding domains that can bind to either the Fc-domain or to the variable heavy chain in the Fab region (1). In biotechnology, SpA is commonly used to purify IgG. In its biological role, SpA binds to IgG to impair phagocytosis by the host’s immune cells (2).

IgG is the most common immunoglobulin in human serum; it is created and released by B-cells and contains two antigen binding sites (3).

Method

Sample preparation

SpA (Sigma) was diluted into labelling buffer (0.2 M NaHCO3 pH 8.3), mixed with Alexa Fluor™ 488 NHS ester (Thermo Fisher Scientific) at a dye-to-protein ratio of 3:1, incubated at 4 °C overnight and purified with a 1 mL Pierce® Desalting Column (Thermo Fisher Scientific) using PBS-T as a buffer.

Anti-EGFR IgG (Absolute Antibody) was used directly from stock as was FreeStyle 293 expression medium (Thermo Fisher Scientific).

Experimental protocol

To obtain binding curves, Alexa Fluor™ 488-labelled SpA at a concentration of 100 nM was mixed with IgG at final concentrations of 0.46 nM - 3000 nM IgG and equilibrated at 4 °C overnight to reach steady state.

Samples were then measured on the Fluidity One-W using the slow flow rate setting, and the KD was automatically generated by a non-linear least squares fitting to Equation 1 (see appendix). Protein-size data collected in the presence of 293 FreeStyle expression medium was corrected by subtracting signal intensities obtained from blank measurements containing 293 FreeStyle expression medium only.

Results

A series of IgG concentrations were added to 100 nM SpA labelled with Alexa Fluor™ 488 in both PBS-T and in FreeStyle 293 expression medium. Binding curves and Rh values of the SpA–IgG interaction in each solution were generated by the Fluidity One-W; from this data the KD was then generated.

ap11 fig 1
Figure 1: Binding curves of IgG against 100 nM Alexa Fluor™ 488-labelled SpA in both buffer and expression medium. All measurements were performed in triplicate. KD was determined by non-linear least squares fitting the data with Equation 1 setting the stoichiometry parameter to n = 3. The KD for the interaction in PBS-T was 47 ± 11 nM and 54 ± 12 nM in FreeStyle 293 expression medium.

Figure 1 shows the binding curves for SpA–IgG complex in both PBS-T buffer and FreeStyle 293 expression medium. Since the average number of IgG molecules bound to SpA had been determined to be three (see separate application note (4) for more details), n = 3 was selected as the stoichiometry parameter in Equation 1.

A fit of the two binding curves in terms of Equation 1 yields a KD value of 47 ± 11 nM in PBS-T and 54 ± 12 nM in the presence of FreeStyle 293 expression medium. The Rh of the SpA–IgG complex in PBS-T was 11.46 nm and 11.97 nm in FreeStyle 293 expression medium. The KD values obtained agreed with the literature (3).

The Rh of free and bound SpA was the same when measured in either PBS-T or FreeStyle 293 medium, indicating that the protein was stable and functional under both conditions.

Conclusions

The binding affinity of SpA and IgG in well defined and complex backgrounds was measured with the Fluidity One-W - with no significant difference in the KD measured. The Rh of the SpA–IgG complex was also found to be the same in both solutions with high precision.

The Fluidity One-W provides binding affinity and precise hydrodynamic radius data in both well defined and complex backgrounds, allowing researchers to avoid lengthy purification steps.

Appendix notes available in PDF below.

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