Bio-Layer Interferometry (BLI) Analysis
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Bio-Layer Interferometry (BLI) provides real-time, label-free measurement of molecular interactions, with a flexible, high-throughput workflow well suited to affinity and kinetic characterization of monoclonal antibodies, proteins, and other biomolecules.
BLI enables parallel analysis across multiple samples with minimal sample handling, making it well suited for rapid screening, affinity ranking, and kinetic profiling. Combined with CovalX’s mass spectrometry-based characterization portfolio, BLI provides complementary interaction data to support biophysical characterization and biotherapeutic development.
How BLI Works
BLI measures binding at the tip of an optical biosensor. One binding partner, the ligand, is immobilized on the sensor tip, and the sensor is then dipped into a well containing the analyte in solution. White light is directed through the sensor and reflects from two surfaces: an internal reference layer and the biolayer at the tip. When the analyte binds to the immobilized ligand, the optical thickness of the biolayer increases, shifting the interference pattern of the reflected light. The instrument tracks this wavelength shift in real time.
Because the sensor moves between sample wells rather than the sample flowing across the sensor, BLI operates without microfluidics. There is no fluidic system to clean between runs, and multiple sensors can be processed in parallel across a microplate. Depending on the assay and sensor chemistry, sensors can be regenerated for reuse or replaced between measurements.
Association rate (ka), dissociation rate (kd), and equilibrium dissociation constant (KD) can be determined from the association and dissociation phases recorded as the sensor moves between analyte and buffer wells.
This open, dip-and-read format makes BLI particularly well suited to high-throughput screening, affinity ranking, and kinetic characterization. Combined with orthogonal analytical techniques such as mass spectrometry, BLI provides complementary information on molecular interactions and supports comprehensive biophysical characterization.
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BLI applications
Antibody screening and drug discovery.
BLI screens large antibody or compound panels against unlabeled antigen, ranking binders early in the campaign. Epitope binning by BLI groups monoclonal antibodies by shared or distinct binding sites on the same antigen, ahead of higher-resolution epitope mapping.
Kinetic and affinity ranking.
The time a lead remains bound to its target, reflected by the off-rate, can inform candidate selection and optimization. BLI provides high-throughput measurement and ranking of binding kinetics and affinity across candidate panels, including ka, kd, and KD.
Target and compound characterization.
BLI characterizes target-compound interactions in real time. It can assess interactions across a range of targets and compounds, supporting hit identification, binding validation, and structure-activity relationship studies.
Comparability and biosimilarity.
Kinetic and affinity profiles generated by BLI serve as a functional fingerprint for comparing production batches, process changes, or innovator and biosimilar pairs, complementing the structural and mass-based comparability data generated elsewhere in the portfolio.
binding affinity measurement 2 BLI deliverables and report structure
| Binding curve data | For each interaction tested, showing association and dissociation phases across the analyte concentration series used. |
|---|---|
| Kinetic and affinity constants | On-rate (ka), off-rate (kd), and equilibrium dissociation constant (KD), calculated from the concentration series by standard kinetic fitting models. |
| Full analytical report | Interpretation of the binding data in a full comprehensive report. |
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Technical notes
CovalX runs BLI analysis on Octet instrument (Sartorius), paired with software for kinetic model fitting. Key parameters:
- Format: open dip-and-read; sensors are moved between microplate wells, with no microfluidics and no fluidic system to clean between runs. This supports parallel processing of large sample panels.
- Sample consumption: typical 50 ug.
- Affinity range and dissociation rate window: 0.1 nM to 1 uM
Every BLI project begins with intact mass spectrometric characterization of the protein by High-Mass MALDI MS, confirming integrity and homogeneity before the interaction experiment.
Buffer composition and sample compatibility are discussed at project setup.
Regulatory Context
Binding kinetics and affinity data generated by BLI support the biological activity and comparability sections of IND, BLA, and biosimilar dossiers.
Frequently Asked Questions
What does BLI measure?
BLI measures the real-time binding interaction between two molecules by tracking the shift in an optical interference pattern at a biosensor tip, as one partner binds the immobilized other. It yields on-rate, off-rate, and equilibrium dissociation constant without requiring a label on either molecule.
How does BLI differ from Surface Plasmon Resonance (SPR)?
Both are label-free, real-time interaction techniques. SPR flows the analyte across an immobilized ligand under microfluidic control and provides higher accuracy at lower sample consumption. BLI dips the sensor directly into the sample with no microfluidics, which supports higher throughput across large panels. Used together, BLI and SPR provide orthogonal, complementary measurements that strengthen interaction characterization across throughput, affinity, and kinetic accuracy.
What sample quantity is required?
Standard projects require 50 µg of each binding protein. Sample type ranges from small molecules to high-molecular-weight proteins.
What deliverables are included in a BLI project?
Clients receive binding curve data, calculated kinetic and affinity constants (ka, kd, KD), and a full written analytical report.
How does BLI relate to CovalX's epitope mapping services?
One CRO, Coordinated Data
BLI analysis at CovalX is integrated with a broader biophysical and mass spectrometry-based characterization portfolio. BLI and SPR provide complementary, orthogonal measurements of molecular interactions, while HDX-MS and XL-MS can further resolve binding interfaces at the molecular level.
Running these analyses through a single CRO reduces sample handling steps, maintains molecular context across techniques, and consolidates orthogonal characterization data into a single deliverable
CovalX Surface Plasmon Resonance (SPR) Services Brochure