Biolayer Interferometry | BLI

Overview

For many molecular interactions it is not only important to know their precise affinity, their thermodynamics, or their stoichiometry. It is often equally important to get detailed knowledge about their underlying kinetics. Two processes constitute the kinetics of a molecular interaction: Association, i.e. the formation of attractive forces between the interacting molecules, and dissociation, i.e. the disengagement of the molecules. In many pharmaceutical and biotech applications, analysis of kinetcs is important, for example to tailor the kinetics of therapeutic or diagnostic antibodies specifically towards rapid association or slow dissociation. In order to derive the kinetics of a molecular interaction, the association and dissociation phases of the interaction have to be monitored over time. A very sensitive, robust, and precise way to do measure binding kinetics is Biolayer Interferometry (BLI). This method detects the build-up of molecular complexes on a sensor-tip, which specifically binds one interaction partner. In simple terms, the BLI setup is an inverse SPR (Surface Plasmon Resonance, Biacore ®) setup.

Loading of one interaction partner ot the sensor tip can be done, for example, with the well-established biotin-streptavidin system. Initially, the sensor tip is dipped into a blank solution (e.g. buffer) to record a baseline (see figure below). Then, one interaction partner (e.g. a biotinylated antigen) is captured on the sensor tip surface and excess antigen is washed off with buffer. In order to monitor the association, the loaded sensor tip is then dipped into a solution containing the second interaction partner (e.g. an antibody). Finally, in order to monitor the dissociation, the sensor tip is dipped into blank buffer solution again, where the ligand (antibody) will dissociate over time from the coupled target (antigen). Certain sensor tips can also be regenerated and re-used for several experiments. Finally, fitting of the association and dissociation phases then provides the respective kon and koff rates and thus kinetics information.

 

BLI, Biolayer Interferometry, Kinetics, SPR, Biacore

Importantly, the Octet® BLI system is label-free and does not require the modification of the interaction partners with fluorescent dyes for example. However, immobilization of one interaction partner has to be accepted in turn. The Octet® BLI system is widely used for quantifying and analyzing the kinetics of molecular interactions involving small molecules, peptides, DNA and RNA molecules, proteins, antibodies, up to whole viruses and bacterial cells. In contrast to the Biacore® SPR system, BLI has the advantage that no microfluidics are required, which makes the system more robust and allows for measuring in complex biological liquids like cell lysate, serum, blood plasma, urine, sea water, etc.

Technology

The Octet® K2 BLI platform is a well-established tool for the kinetics analysis and characterization of molecular interactions between proteins, nucleic acids, and small-molecules. It is also widely used for quantification experiments as well as in drug discovery for kinetic screenings and kinetic optimization.

The Octet® K2 BLI platform is based on the optical and label-free analytical technique called Biolayer Interferometry (BLI). BLI analyzes interference patterns of white light that is reflected from two optical layers of a very small (600 µM diameter) tip: One internal reference layer inside the tip and one layer at the interface between the tip and the surrounding liquid (Figure below, panel A). Each reflection generates constructive and destructive interferences that vary with the wavelength (Figure below, panel B, gray curve). Any change at the outer layer of the tip (a biocompatible surface with one interaction partner immobilized on it), for example due to binding of a ligand, leads to different interference patterns at this reflective layer. This, in turn, causes a shift of the interference spectrum to different wavelengths (Figure below, panel B, red curve). From the time-resolved monitoring of this shift, it is possible to derive real-time association and dissociation rates of the ligands in solution to the immobilized interaction partner at the tip surface.

BLI, Biolayer Interferometry, Kinetics, SPR, Biacore

BLI allows for the real-time determination of the interaction dissociation constant (KD), as well as the observed association (kon) and dissociation (koff) rate constants. A wide range of different sensor tip surfaces allows the precisely tailored immobilization of one interaction partner. Common techniques for immobilization are direct surface immobilization using amine-reactive coupling, biotin-streptavidin based coupling, anti-GST– or anti-histidine-tag based coupling, and antibody-based coupling. Thus, BLI using the Octet® system generates a complete kinetic profile of a molecular interaction and by that, for example, help with a more-fine grained analysis of molecular interactions with similar equilibrium affinities.

Here at 2bind, the state-of-the-art the PALL FortéBio Octet® K2 BLI System is used. This BLI system features two parallel measurement channels, a 96-well sample plate format, excellent sensitivity down to analytes of 150 Da, and high reproducibility. A sample consumption of only 200 µL and the possibility to re-use a sample for multiple measurements enables that analysis of precious and difficult to obtain analytes. The Octet® K2 system features a dynamic range of association and dissociation rate constants of six orders of magnitude (kon: 101 – 107 M-1s-1, koff: 10-6 – 10-1 s-1). Affinities (KD) can be determined in the range from 10-3 – 10-11 M. The lower and upper boundaries for quantification are 0.05 µg/mL and 2000 µg/mL, respectively.

Typical applications

The versatile and robust BLI system offers a wide variety of different experiment possibilities with respect to kinetics, quantitative, screening, and assay development applications:

Kinetics applications

  • Protein-protein interactions
  • Antibody characterization
  • Antibody-antigen binding
  • Protein-small molecule interactions
  • DNA-aptamer binding
  • Bacteria-antibody interactions
  • Virus-like particle-antibody/protein binding
  • GPCR-protein binding

Quantitative applications

  • Antibody quantitation
  • ELISA replacement
  • Protein quantitation in crude extracts
  • Detection of contaminations

Screening applications

  • DNA-aptamer screening
  • Small molecule fragment screening (with certain limitations)
  • Secondary screening and hit validation (with certain limitations)
  • Inhibitor screening

Assay development applications

  • Formulation development
  • Media development

For examples of such applications, take a look at our Application Database.

Advantages

  • Label-free detection method no fluorescent dyes required
  • Free choice of assay buffers  also gylcerol or DMSO possible as solvents
  • Complete kinetic characterization  association and dissociation rate constants determinable
  • Numerous coupling methods covalent and affinity-based coupling methods available
  • Measure at elevated temperatures analysis possible up to 40°C
  • Measure in crude biological liquids cell lysate, serum, plasma, environmental samples
  • Wide range of binding affinities range from nM to mM affinities measurable
  • Wide range of possible molecules from large VLPs to, under certain conditions, small molecules

FAQ – General

What is BLI?

BLI or “Biolayer Interferometry” is a label-free biosensor technology that allows for real-time analysis of the kinetics of molecular interactions by detecting changes in interference patterns of white light reflected from the surface of fiber-optic sensor tips.

What parameters of a molecular interaction can be determined with BLI?

BLI directly enables the real-time determination of association rate (kon) and dissociation rate (koff) of a molecular interaction; thus its kinetics. From these rates, the overall affinity of the interaction (KD) can be calculated. The KD value can also directly be determined from the concentration-dependent formation of molecular complexes.

If you are only interested in the steady-state affinity of a molecular interaction, consider using MicroScale Thermophoresis as an alternative. This method allows for the highly precise, robust, and fast determination of the dissociation constant of molecular interactions. If you are interested in the thermodynamic parameters of an interaction, take a look at Isothermal Titration Calorimetry.

What is the slowest dissociation rate that can be measured?

The primary limitation for the measurement of very low dissociation rates is sample evaporation from the microplate wells.

What is the fastest association rate that can be measured?

The primary limitation for the measurement of very high association rates is the data-sampling rate. In practice, association phases can be measured that reach their plateau in as little as 20 seconds.

Why does the vertical axis of BLI plots display a thickness in nm?

BLI measures the changes in interference patterns of white light at the surface of a fiber-optic sensor tip. These changes in turn are a function of changes in the thickness (in nm) of the molecular layer on top of the sensor tip surface.

FAQ – Samples

How much sample is required for BLI assays?

With the Octet® K2 BLI system and standard 96-well plates, a minimum sample volume of 180 µL is required in order to assure sufficient orbital flow around the tip. Importantly, the sample can be fully recovered from the plate after the measurement, because the sample is analyzed non-destructively. Sample volumes below 180 µL are not recommended, as they may lead to internal reflections in the 96-well plate.

If these requirements are problematic for your special case, MicroScale Thermophoresis is a great alternative to determine steady-state affinities of molecular interactions with minimal sample consumption.

What concentration should samples have?

The specific sample concentration required for precise measurements depends on sample type, interaction affinity, as well as the assay buffer or solution. For example, human IgG antibodies can be analyzed in concentrations between 50 ng/mL and 2000 µg/mL.

Is it possible to analyze complex and crude samples?

For most biological applications, BLI measurements are independent of the buffer composition or the composition of the sample medium. For example, small-molecules can easily be analyzed in buffers with high DMSO-concentrations. However, for many small molecules, potential influence of the biological liquids on detection sensitivity has to be considered. Moreover, complex biological liquids like serum, cell fractions and cell lysates, as well as bacterial lysates or cell culture supernatants can be analyzed.

FAQ – Assay Conditions

Which types of sensor tips are available?

Around 20 different sensor types are available, including sensors coated with streptavidin for capturing biotinylated ligands, specifically coated sensors for capturing all kinds of antibody molecules and fragments, sensors with amine-reactive coating, affinity sensors against His- or GST-tags, as well as sensors coated with protein A, protein G, or protein L. A full list of available sensor types is available from PALL FortéBio.

How many binding sites are on the surface of a sensor tip?

PALL FortéBio sensor tips feature a base protein coating in order to assist in reproducible ligand capturing and to minimize non-specific binding. The sensor tips approximately provide 109 capture sites.

Is non-specific binding a problem?

In general, the sensor tips from PALL FortéBio cotain a base protein layer on their surface that minimized most non-specific binding. This layer also enhances ligand capture by preventing them from directly contacting the sensor surface. If further reduction of potential non-specific binding is required, this can usually be achieved with blocking steps with BSA for example.

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