BLI vs. SPR: Choosing the Ideal Method for Analyzing Biomolecular Interactions

Understanding the kinetics of molecular binding or measuring the concentration of active analytes are fundamental characterization methods, both in basic research and drug discovery. Unlike endpoint methods such as ELISA, real-time, label-free techniques for analyzing biomolecular interactions do not require labeling any of the interactants, allowing for the design of simpler assays. The lack of fluorescent labels and/or secondary detection reagents also eliminates interference or related experimental artifacts, leaving you with clean data that better captures biologically relevant interactions.

Biolayer interferometry (BLI) and surface plasmon resonance (SPR) provide real-time data and are valuable for understanding biomolecular mechanisms, offering insights into the kinetics, affinity, and specificity of interactions. The choice between BLI and SPR depends on the specific study requirements, such as sensitivity, throughput, and sample type.

BLI operates without fluidics, using light interference to detect molecular binding, making it suitable for unpurified biological samples and high-throughput screening. Since purified biological samples are not required and cell lysates can be used, interactions can be measured under native conditions.

SPR detects mass changes associated with refractive index shifts, offering high sensitivity for protein interactions and drug-active substances in purified samples.

What is Bio-layer Interferometry?

Bio-layer Interferometry is an analytical technique that monitors the interference pattern of white light reflected from two surfaces: the first surface is the layer of immobilized protein on the tip of a biosensor, and the second is an internal reference layer.

BLI technology operates by moving the biosensor to a 96- or 384-well plate and "dipping" it into the sample (known as dip-and-read), providing a robust and straightforward way to introduce the analyte to the sensor surface. When an analyte binds to the immobilized ligand, BLI detects a change in the thickness of the biolayer on the biosensor tip by measuring the interference between light waves, or wavelengths.

Octet^® BLI analytical systems enable real-time, label-free analysis of biomolecular interactions—used to determine kinetics, affinity, and antibody/protein quantification with unprecedented time and cost savings. Applications of Octet^® BLI include:

• kinetics of binding: protein-protein interactions, antibody characterization, antibody-antigen binding, DNA-aptamer, particle-antibody, bacteria-antibody interactions, and more
• antibody quantification: quantitative protein determination, AAV titer, vaccine-induced antibody responses, potency tests, immunogenicity, contamination detection, and glycosylation profiling
• screening: binding screening, DNA-aptamer and inhibitor screening, clone selection
• assay development: formulation development, media development, cell line development and optimization, test validation

Time and Cost Savings with Octet® BLI Technology:

• higher throughput enables protein quantification in just two minutes and the parallel processing of up to 96 samples
• automated analysis for faster results and quicker time-to-market
• real-time analysis of molecular interactions
• measurement of analytes in unpurified samples, such as cell cultures, lysates, or supernatants
• ease of use with an integrated user interface designed for biologists
• fluidics-free system simplifies direct analysis in early research stages and is low-maintenance

What is Surface Plasmon Resonance (SPR)?

Early identification of kinetics, affinity, and competitive properties of compounds is crucial for making informed decisions in the early stages of drug discovery. Surface plasmon resonance (SPR) is an analytical method for measuring molecular interactions in real-time.

SPR technology utilizes a continuous flow system to deliver analytes to an immobilized ligand on a biosensor chip. Molecular binding of the analyte to the ligand causes a change in mass and refractive index on the sensor surface, resulting in a shift in the reflective index that can be observed on a sensorgram.

The Octet^® SPR device includes a biosensor, consisting of a chip made from a thin microscope slide coated with a thin, semi-transparent gold film. This setup enables light reflection and generates a surface plasmon wave at the interface between the sample and the gold surface at a specific angle. Changes in the refractive index allow for the detection of molecular interactions. SPR tests often involve a flow-based fluidics system to deliver analytes to the biosensor surface. Typical binding assays, whether equilibrium-based or kinetic, require titration of multiple analyte concentrations to accurately determine kinetic and affinity constants.

While traditional SPR binding platforms use fixed concentration injections (FCI) to deliver samples, the Sartorius Octet^® SF3 SPR platform offers "Next Generation" technology through OneStep^® and NeXtStep™ injections.

Accurate and rapid biological discoveries with Octet® SPR technology

• advanced gradient injections for precise measurement and assessment of molecule kinetics and affinity from a single analyte concentration in a single assay
• use of two buffers during a single analysis
• optimized fluidic system minimizes potential clogging sources with effective decontamination protocols
• sample injection for recovering bound analyte allows for the conservation of valuable analyte
• temperature control enables the evaluation of kinetics and affinity of therapeutics at physiological temperatures.

High-Throughput sample collection

• generate complete kinetics and affinity data for up to 768 samples in a single assay
• OneStep^® Injection Technology: more than 72 hours of unattended operation and a unique sample scheduling system enable high-throughput collection and analysis of hundreds of samples in a single cycle

OneStep® Injection Technology

• saves time, reagents, and plate space, while reducing potential errors in assay development
• enables precise measurement of binding kinetics and affinity from a single analyte concentration

NeXtStep™ Gradient Injections

• precise competitive assays enable the examination of analyte behavior and the presence of competing molecules from a single injection
• assessment of multiple analytes and competitors in a single test, without the need for the competitor to be present in the liquid buffer

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The best technique choice

Choosing between BLI and SPR techniques depends on the application and the size range of the molecules involved. SPR, for example, is a more sensitive technique for measuring interactions with small molecules and fragments. On the other hand, BLI is better suited for high-throughput analysis in the pharmaceutical industry, offering additional flexibility for testing unpurified biological samples, such as cell lysates, during process optimization or bioprocess monitoring.

While these technologies are quite powerful individually, together they’re a winning combo in terms of answering the widest range of questions.

Complementary approach

Sartorius is the only provider of both industry-leading technologies under one trusted brand. The fluidic-free Octet^® BLI range is synonymous with speed and ease of use, while the Octet^® SPR range has patented injection tools that simplify multi-concentration studies.

When used orthogonally, BLI and SPR are powerful, complementary tools for characterizing biomolecules, such as antibodies, fragments, or other biotherapeutic proteins, in research and workflow analyses. This orthogonal, complementary approach is particularly useful for workflows involving initial screening, such as direct screening of supernatants or phage display in antibody development. Together, these techniques enhance the robustness of biomolecular interaction studies, optimizing resource use and saving time and costs.

In A Compendium for Successful BLI and SPR Assays, find detailed chapters and expert advice on designing and conducting assays to measure the kinetics of biomolecular interactions.

In conclusion, both BLI and SPR provide real-time analysis of molecular interactions, aiding in the understanding of binding mechanisms. The choice of the right platform depends on the analysis objectives, sample types, and specific laboratory factors. Each technique offers unique advantages that address different aspects of drug discovery processes.

The text was downloaded from the the official Sartorius website.

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