Your Antibody Barely Dissociates. How Much Can You Trust the Off-Rate?

An antibody binds its target. The dissociation phase starts. The curve barely moves.

That can be encouraging. It also raises a question I keep returning to as I learn more about surface plasmon resonance, or SPR:

Did we observe enough dissociation to rank this antibody confidently?

A small off-rate needs a convincing curve

SPR follows binding and dissociation in real time. The off-rate, written as koff, describes how quickly a bound complex separates.

A smaller value means slower dissociation under the conditions tested. The Assay Guidance Manual explains how these rates relate to binding affinity.

Consider a hypothetical interaction with koff = 0.00001 s⁻¹. Under a simple 1:1 model, the calculated loss of binding-specific signal after ten minutes is only about 0.6%.

That is a small change to build a candidate ranking around.

The question becomes whether the assay can distinguish that movement from background drift, surface instability and measurement variability.

Check what is keeping the signal on the surface

Before trusting a very slow off-rate, I would look at three things:

• The background. Reference and buffer-blank measurements help establish whether the small signal change belongs to the interaction. The captured material also needs to remain stable. Research on SPR surface design shows how loss of captured protein can complicate slow-dissociation measurements.
• The binding arrangement. A molecule can detach and bind again nearby. An antibody can also engage through both binding arms, creating avidity: the combined strength of multiple contacts. Rebinding and bivalent binding can both slow apparent dissociation.
• The consistency. Repeat the measurement, inspect the gaps between the data and fitted curve, and check whether a longer dissociation window supports the same result. Close candidates need differences large enough to stand above the assay’s variability.

A smooth fit becomes much more useful when the assay supports its assumptions.

Temperature can reveal more of the interaction

This is one reason I find the temperature flexibility of Alto Digital SPR useful.

In Nicoya’s temperature-dependence study, the tested antibody–antigen pair dissociated approximately 5.6 times faster at 37°C than at 25°C. Its measured affinity also weakened. These are manufacturer application data.

That gives a scientist another useful measurement condition, especially when 37°C fits the biological question.

The temperature belongs with the result. A value measured at 37°C describes that condition; it cannot replace an unresolved off-rate at 25°C. Candidate rankings should be compared under matched conditions.

Equilibrium fitting gives you another route to affinity

Alto also supports steady-state affinity fitting.

This approach uses the binding response across concentrations after equilibrium has been reached to estimate KD, the equilibrium dissociation constant. It provides another route to affinity without requiring separate estimates of both rate constants.

The requirement is real equilibrium across an informative concentration range. Tight-binding interactions can take a long time to get there, particularly at low concentrations, as Jarmoskaite and colleagues demonstrate.

Equilibrium fitting can answer how tightly the partners bind. It does not, by itself, establish how quickly they separate.

Make confidence practical

Nicoya lists 16 parallel sensors, automated on-cartridge serial dilutions and sample volumes starting at 2 µL for Alto.

What interests me is the room that creates for assay development: allocating material to replicates, checking surface loading and comparing candidates under matched conditions, with less manual dilution work.

Before advancing a candidate on a tiny fitted off-rate, ask whether its ranking survives those checks. If two slow binders remain unresolved, report that clearly and choose the next experiment that can separate them.

The goal is a candidate ranking you can defend with the measured data.

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Desmond Gilmour

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I work at Nicoya Lifesciences. The views expressed here are my own and do not necessarily reflect those of Nicoya. This is a personal publication, not an official company communication.