Formula used
Independent sites
P(i) = C(n,i) θi(1 − θ)n−i
Independent sites share one affinity and do not communicate. State probabilities follow a binomial distribution. The mean bond count is probability weighted.
Binding polynomial
P(i) = βᵢ[L]ⁱ / Q
Sequential Kd values produce cumulative association constants. Adair mode accepts those cumulative constants directly. Every state weight is normalized by the binding polynomial.
Hill model
The Hill coefficient controls empirical steepness. It does not necessarily equal the physical site count. Mechanistic interpretation requires independent evidence.
Avidity approximation
Avidity factor = Bv−1
Kdapp = Kdcompetition / Avidity factor
G combines distance, linker, orientation, steric, activity, and accessibility factors. S represents density, contact area, and mobility. I is an intramolecular correction. This transparent expression is phenomenological, not universal.
Competition
This equation models simple competition at the same site. Multivalent competitors may require a larger state model. Competitor valency remains documented for extension.
Kinetics
Association = θeq(1 − e−kobst)
Dissociation = Signal₀e−koff,efft
Rebinding reduces the effective dissociation rate. Internalization and degradation add irreversible losses. Full transport and state-transition kinetics need specialized models.
Free energy and mass balance
[L]total = [L]free + [R]total⟨bonds⟩
Kd is converted to molar units before free-energy calculation. The mass-balance equation is solved by bisection. This matters near stoichiometric conditions.
How to use this calculator
- Select the simplest model compatible with available data.
- Enter total ligand and receptor concentrations with correct units.
- Set ligand and receptor valencies.
- Use a monovalent intrinsic Kd whenever possible.
- Configure effective concentration, linker reach, spacing, orientation, and sterics.
- Add receptor density, contact area, and mobility for surface systems.
- Enable a competitor only when it shares the relevant site.
- Use exact mass balance when receptor depletion can matter.
- Run uncertainty analysis with realistic coefficients of variation.
- Compare models and inspect warnings before interpretation.
- Fit experimental data as an empirical cross-check.
- Export CSV, JSON, or a print-ready PDF report.
Interpretation and limitations
Affinity and avidity
Affinity describes one interaction. Avidity reflects multiple engagements, rebinding, geometry, and presentation. Apparent Kd therefore depends on the selected model and assay context.
State probabilities
State zero is unbound. Higher states represent more simultaneous engagements. The full state is the maximum engagement allowed by the smaller valency.
Superselectivity
The density selectivity alpha is a local logarithmic slope. Values above one indicate sharper-than-proportional density response. The value changes across receptor-density ranges.
Uncertainty
Monte Carlo intervals include only selected parameter variation. They exclude unmodeled structural and measurement uncertainty. Use experimentally justified distributions.
Optimization
The optimizer searches bounded valency and linker grids. It rewards target binding and selectivity while mildly penalizing complexity. The result is exploratory rather than manufacturing guidance.
Frequently asked questions
Which Kd should be entered?
Enter a monovalent intrinsic Kd when available. Entering an avidity-enhanced Kd may double count multivalent effects.
Why is apparent Kd extremely small?
High effective concentration, favorable geometry, density, cooperativity, and valency multiply. Review each assumption and compare against experimental data.
Does Hill coefficient equal valency?
No. It is mainly an empirical slope parameter.
When is exact mass balance important?
Use it when receptor concentration is not negligible relative to ligand. It is especially relevant near stoichiometric conditions.
Can this fit SPR or BLI sensorgrams?
It provides a compact kinetic approximation and an equilibrium Hill fit. Full sensorgram analysis may require transport, heterogeneity, and rebinding models.
Can heteromultivalent systems be modeled?
The current state engine uses one effective receptor class. Add receptor-specific state weights and affinity matrices for a fully heterogeneous system.
Is the optimizer definitive?
No. It is a bounded exploratory search based on the selected phenomenological equations.
Can results be saved as PDF?
Use Print or PDF, then select the browser save-as-PDF destination.
Is this suitable for clinical decisions?
No. Validate equations, parameters, and numerical behavior before regulated, diagnostic, or clinical use.