How Biolayer Interferometry enables label-free kinetic analysis of antibody-antigen interactions
Imagine a microscopic world inside your body, where a constant, silent war is waged against invaders like viruses and bacteria.
The elite special forces in this war are antibodies—Y-shaped proteins that seek out and neutralize specific enemies, called antigens. The moment an antibody grabs hold of an antigen is a critical event, a molecular handshake that determines the success of an immune response. But how can we see this event? How can we measure how tight the grip is, or how long it lasts?
For decades, scientists struggled to observe these interactions without interfering with them. It was like trying to measure a handshake by gluing sensors to the participants' palms—the process itself changed the outcome. This changed with a revolutionary technology called Biolayer Interferometry (BLI), a molecular stopwatch that lets us watch this dance in real-time, without any labels . This isn't just academic curiosity; it's the cornerstone of developing new vaccines, cancer therapies, and diagnostic tests faster and more effectively than ever before .
At its heart, BLI is an elegant and simple concept: it uses light to measure what's happening on a molecular scale.
Think of it like this: you dip the tip of a special biosensor—essentially a tiny, clear fiber optic wire—into a solution. This tip is coated with a "bait" molecule, say, an antibody. You then shine a beam of white light down the sensor. Some of this light reflects off the inner surface of the tip, and some reflects off the outer surface where your bait molecules are. These two reflected beams travel back and combine, creating an interference pattern.
When the tip is bare, the interference pattern has a specific signature.
When an antigen from the solution binds to the antibody on the tip, it adds a tiny but measurable layer of mass. This changes the distance the outer light beam travels, which in turn shifts the interference pattern.
By tracking these shifts in the interference pattern in real-time, the BLI instrument creates a direct, label-free movie of the binding event. It tells us not just if molecules bind, but how quickly they associate and how slowly they dissociate—the very essence of their interaction .
Let's dive into a crucial experiment where a pharmaceutical company needs to test a new monoclonal antibody drug (mAb) designed to target a protein (antigen) on a cancer cell.
The entire experiment is automated and happens in a small tray containing tiny vials of solution. The process can be broken down into five key steps:
A biosensor tip coated with a protein that captures our antibody drug is dipped into a simple buffer solution.
The tip is moved to a vial containing the new antibody drug. The drug molecules attach to the capture protein.
The tip goes back into the buffer to wash away any loose molecules and stabilize the signal.
The tip is dipped into a vial containing the cancer antigen. We watch in real-time as binding occurs.
The tip is moved back to plain buffer to observe how quickly the antigen falls off.
A sample sensorgram, the movie of the binding event, showing the five key steps of the BLI assay.
How the binding signal increases with higher antigen concentrations.
| Antigen Concentration (nM) | Max Response (nm) | Steady-State (nm) |
|---|---|---|
| 0 (Control) | 0.05 | 0.05 |
| 10 | 0.45 | 0.40 |
| 25 | 0.98 | 0.85 |
| 50 | 1.60 | 1.35 |
The kinetic constants reveal the quality of the interaction.
| Parameter | Symbol | Value |
|---|---|---|
| On-rate | kᵒⁿ | 2.5 × 10⁵ M⁻¹s⁻¹ |
| Off-rate | kᵒff | 1.0 × 10⁻⁴ s⁻¹ |
| Affinity | KD | 0.4 nM |
Why kinetics matter in drug candidate selection.
| Antibody Candidate | kᵒⁿ (M⁻¹s⁻¹) | kᵒff (s⁻¹) | KD (nM) | Verdict |
|---|---|---|---|---|
| mAb-A (Our Drug) | 2.5 × 10⁵ | 1.0 × 10⁻⁴ | 0.4 | Excellent |
| mAb-B | 5.0 × 10⁵ | 1.0 × 10⁻² | 20.0 | Reject |
This comparison highlights why kinetics matter. Even though mAb-B binds faster, its rapid dissociation makes it a poor drug candidate. BLI provides the critical data to make this informed choice early in development .
Essential reagents for a successful BLI experiment.
The core of the system. These disposable tips have specialized surfaces to capture the "bait" molecule reliably.
The molecule attached to the biosensor that grabs your protein of interest (e.g., Protein A).
The pair of interacting molecules. The Ligand is immobilized, and the Analyte is in solution.
A carefully formulated solution that mimics a biological environment to keep proteins stable.
A mild solution that breaks the antibody-antigen bond without damaging the biosensor.
Reference materials to validate assay performance and ensure data quality .
Biolayer Interferometry has transformed the way we study molecular interactions.
By providing a label-free, real-time, and quantitative look at the fundamental dances of biology, it has removed a major bottleneck in drug discovery and biomedical research. It allows scientists to rapidly screen thousands of potential drug candidates, selecting only the most promising ones—those with the perfect kinetic grip—to move forward into clinical trials.
This not only saves billions of dollars but, more importantly, accelerates the journey of life-saving therapies from the lab bench to the patient's bedside. In the unseen war within our bodies, BLI is the powerful spyglass that gives our side the ultimate advantage .
No fluorescent or radioactive tags needed
Watch interactions as they happen
Precise kinetic and affinity measurements