Bio-layer interferometry (BLI)

BLI is an efficient tool for characterizing interactions between various classes of biomolecules and is often seen as the high-throughput alternative to SPR. Commercially introduced 15 years ago its popularity as a biosensor technology grew rapidly. It makes it possible to determine kinetic rate constants and the binding affinities of molecular interactions, without the need for labels.³⁶ BLI is similar to SPR in the sense that both require immobilization of a ligand on a sensor surface, where analyte binding is detected using an all-optical method (Fig. 2d). In BLI, white light is directed through an optical fiber to a biocompatible layer on the fiber surface. Once ligand molecules are immobilized on this biolayer, the fiber becomes a probe which can be dipped into analyte solution.

Analyte binding changes the refractive index of the biocompatible layer, which is measured as a change in interference pattern and is proportional to the amount of bound analyte. The interference arises from the small path difference between light reflected from the fiber surface/biolayer interface and the biolayer/solution interface.³⁷

In contrast to SPR, where the binding surface is exposed to a continuous flow, the BLI sensor tip is dipped in static solutions of ligand and repeated for a series of dilutions. The use of orbital agitation of the sample holder at high speed ensures sufficient mixing to minimize mass transport limitations. With this approach, association and dissociation of the analyte and binding partner can be followed over time to extract k_on and k_off (Fig. 2e) and binding affinities in the range of 10 pM to 1 mM are accessible. In many cases, a 1 : 1 interaction model is applied to fit the model to experimental data.^38,39

Given that experiments can be performed in micro-well plates, no maintenance-intensive microfluidics are needed. Setups can be combined with disposable sensor tips, which enables coupling to 96- or even 384-well high-throughput formats.^40,41 However, the advantage of higher-throughput comes with inferior reproducibility and lower sensitivity to low MW analytes, so cross-validation by SPR is generally recommended.⁴⁰

Similar to SPR, BLI has limitations due to mass transfer and immobilization effects.³⁶ A discussion of immobilization effects can be found in the work of Kamat et al. where they developed a binding kinetic assay to quantify antigen–antibody interactions.⁴² Advances in data analysis should further help to go beyond the often assumed 1 : 1 interaction models.^43,44

Among recent applications since 2019 are several studies focusing on SARS-CoV and MERS-CoV interactions. In Fig. 2f, Yi et al. use BLI to compare binding affinities of full-length human ACE2 (blue) to the receptor binding domain (RBD) of SARS-CoV-2 (orange).²³ Data was recorded on an Octet RED96 instrument at different concentrations (1.85 nM (black), 5.56 nM (green), 16.67 nM (blue), 50 nM (red)). Binding kinetics were evaluated with a 1 : 1 Langmuir binding model by ForteBio Data Analysis 9.0 software and yielded k_on = 2.65 × 10⁵ M⁻¹ s⁻¹, k_off = 1.35 × 10⁻³ s⁻¹ and K_d = 5.1 nM.

In similar studies, BLI has been used to study the spike glycoprotein (HIV-1, SARS-CoV-2, MERS-CoV) and its binding to receptors and antibodies,^22,45–49 including antibody competition assays for humoral protection.^50,51 BLI has also been used to quantify cross-reactivity of antibodies for Nipah virus (NiV) and Hendra virus (HeV), against which no vaccines or licensed therapeutics exist yet.⁵² Li et al. made use of the concentration-dependent on-rates of antigens binding to immobilized antibodies to quantify antigen levels in a high-throughput manner in CHO cell line development. They compared BLI's sensitivity and throughput to widely-used assay formats, such as ELISA.⁵³ A similar approach was used by Wallner et al. to quantify glycosylation of Fc-glycosylated IgGs via immobilized lectins as measure for product quality.⁵⁴ Loomis and Steward-Jones et al. used BLI to evaluate the antigenicity of their candidate vaccines in structure-based design of Nipha virus vaccines.⁵⁵

In summary, BLI is widely used for binding affinity and kinetics measurements of protein–protein interactions, and is increasingly applied as a complementary method to SPR.