Competition Assays vs. Direct Binding Assays: How to choose 

Drug discovery and biomolecular research often come down to a simple question: how well do these two molecules interact? There are two major ways to answer that question – direct binding assays and competition (displacement) assays – and each provides different kinds of information about affinity, mechanism, and potency.

If you’re deciding which approach to use, this guide walks through what each assay measures, when to choose one over the other, and how new microfluidic technologies make the decision easier than ever.

1. What is a direct binding assay?

A direct binding assay measures the interaction between a target and a ligand without a competitor. In other words, direct binding tells you how tightly and how quickly two molecules interact.

Primary readout:

The readout comes from a physical change that occurs when the complex forms-mass, fluorescence, heat, or hydrodynamic size. This makes direct assays ideal for determining:

• • true affinity (K_D)
  • kinetics (K_on/K_off) or stoichiometry [1] (in some methods)

Common formats of direct binding assays [1-4]:

• • Surface-based (SPR, BLI): One partner is immobilized, and binding is measured through real-time association/dissociation.
  • Solution-based (ITC, MST, MDS): Both partners remain free in solution, and binding is detected through heat, mobility, or size changes.

2. What is a competition assay?

A competition assay measures how effectively an unlabelled test ligand can displace a labelled reference ligand (tracer) from a target binding site [5].

The tracer binds the target to form a complex that produces a measurable signal, such as a change in fluorescence or, in the case of microfluidic diffusional sizing (MDS), a shift in hydrodynamic radius as the complex diffuses more slowly than the free tracer [4, 6]. As increasing amounts of test ligand are added, it competes with the tracer for the same site, reducing the amount of tracer–target complex.

Because the test ligand remains unlabelled and fully in solution, competition assays are especially valuable when modification or immobilization would disrupt binding, or when high-throughput Structure–Activity Relationship (SAR) screening requires rapid comparison of many related molecules under identical conditions [5, 7].

Primary readout:

Competition assays yield an IC₅₀: the concentration of test ligand that displaces 50% of the tracer. IC₅₀, however, is not an intrinsic affinity because it depends on the tracer concentration and how tightly the tracer binds [5, 7]. To obtain the true inhibition constant (Kᵢ), IC₅₀ must be interpreted using the Cheng–Prusoff relationship, which accounts for these parameters [8].

In tight-binding situations, where inhibitor and target concentrations are similar, the Cheng–Prusoff correction breaks down, and Kᵢ must instead be determined using the Morrison equation, which properly accounts for ligand depletion [9].

Important practical limitations [5, 7, 9, 10]

• • K_i cannot be calculated unless the tracer Kᴅ is known.
  • Assumes competitive, reversible, single-site binding at equilibrium.
  • Allosteric or non-competitive mechanisms break the IC₅₀ → Kᵢ relationship.
  • Tight-binding systems require the Morrison equation rather than Cheng–Prusoff.
  • Tracer affinity and stability strongly influence assay sensitivity and accuracy.
  • IC₅₀ values are assay-dependent and not directly comparable across setups.

3. Direct vs. competition: a practical decision guide

Selecting between a competition assay and a direct binding assay determines not only what kind of data you get, affinity, kinetics, stoichiometry, or rank-order potency, but also how confidently you can interpret it. The decision influences assay development time, throughput, and even whether weak or transient interactions are detected at all.

Choosing between direct and competition assays depends on the question you’re trying to answer and how your target behaves. In practice, most projects benefit from using both approaches at different stages.

Key considerations

A. Mechanistic detail vs. potency ranking

• • Direct binding: true K_D, and in some workflows K_on/K_off and stoichiometry
  • Competition assays: efficient potency ranking across many molecules

If you need depth, choose direct. If you need speed, choose competition.

B. Does labelling or immobilization alter the interaction?

If labelling the test ligand or immobilizing the target affects binding, competition assays avoid the modification entirely.

However, for in-solution methods such as Microfluidic Diffusional Sizing, this constraint is often eliminated as it measures the binding without immobilization.

C. What affinity range are you working in?

• • Weak binders (µM–mM): competition assays with a strong tracer improve sensitivity
  • Very tight binders (nM or tighter): direct assays can be distorted by rebinding or depletion; competition may give cleaner data

D. What level of throughput do you need?

• • High-throughput screening, SAR, fragments: competition
  • Mechanistic studies, confirmations, epitope mapping: direct binding

E. What technologies are available?

Traditionally, this choice has often meant committing to a platform: surface-based technologies for direct binding or tracer-based displacement assays for competition.

However, advances in microfluidic diffusional sizing (MDS) now allow binding interactions to be characterized in free solution by monitoring diffusion-derived hydrodynamic radii, including under native or complex sample conditions [11]. Implemented in instruments such as the Fluidity One-M, MDS enables both direct titrations and competition assays to be performed on the same platform, in the same buffer, without immobilization or extensive re-engineering of assay formats (figure 1) [12].

4. A unified approach with Microfluidic Diffusional Sizing (MDS)

Microfluidic Diffusional Sizing technology run on the Fluidity One-M system eliminates the historical divide between “direct” and “competition” workflows. Both can be run on the same platform, in the same buffer, using the same tracer strategy, so assay design follows biology, not instrumentation constraints.

For direct binding assay: MDS measures direct binding in free solution, without immobilization or surface artifacts. This preserves the molecule’s native state and enables accurate KD determination even for membrane proteins, intrinsically disordered regions, or samples measured directly in complex buffers [4, 13].

For competition assays: Because MDS measurements occur in free solution at equilibrium, MDS allows:

• • reliable determination of tracer Kᴅ through direct binding [10].
  • clean IC₅₀ measurements free of surface and wash artifacts
  • accurate Kᵢ calculations under matched assay conditions
  • seamless transition from competition screening to direct binding validation

Table 1. Comparison of direct binding and competition assay formats. Both approaches can be performed in free solution on the Fluidity One-M, allowing assay choice to follow biological needs rather than instrumentation constraints.

5. Conclusion

Direct binding and competition assays provide complementary insights into molecular interactions. Direct assays reveal true affinity, stoichiometry, and mechanistic detail, while competition assays enable efficient potency ranking: particularly when test ligands cannot be labelled or immobilized.

Traditionally, choosing between these formats required choosing between platforms or accepting compromises in assay conditions. With microfluidic diffusional sizing (MDS) on the Fluidity One-M, both approaches can be performed in free solution, at equilibrium, and on the same instrument, eliminating surface artifacts and reducing sample consumption.

By combining rapid competition measurements with precise direct binding titrations in one workflow, researchers can move seamlessly from early screening to deep mechanistic understanding: letting the biology dictate the assay strategy, not the instrumentation.

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