Ligand Binding Affinity Evaluation

Ligand binding affinity quantifies the strength of interaction between a biomolecule (e.g., protein, antibody, or nucleic acid) and its binding partner (ligand), such as a small-molecule drug, peptide, or cofactor. It is expressed as the equilibrium dissociation constant (K_D), where a lower K_D indicates stronger binding. This parameter is governed by non-covalent interactions - hydrogen bonding, electrostatic forces, hydrophobic effects, and van der Waals interactions - and is critical for understanding molecular recognition, drug-target engagement, and biological function. In biopharmaceuticals, precise affinity measurements ensure therapeutic efficacy, minimize off-target effects, and guide rational drug design.

Equilibrium dissociation constant and its measurement applications diagram. (STEMart Original)

Our Techniques

Technique	Throughput	Kinetic Data	Sample Use	Key Limitation
SPR	Medium	Yes	Low (~μg)	Requires immobilization optimization
BLI	High	Yes	Very low	Limited multiplexing
GCI	Medium	Yes	Low	Specialized instrumentation
ITC	Low	No	High (~mg)	Low sensitivity for weak binders
ELISA	High	No	Medium	Semi-quantitative, endpoint only

1. Surface Plasmon Resonance (SPR)

SPR detects real-time biomolecular interactions by measuring refractive index changes near a sensor surface. Ligands are immobilized on a chip, and analytes flow over them. Binding events alter plasmon resonance angles, generating sensorgrams to calculate K_D, association rate, and dissociation rate.

The gold standard for label-free, kinetic profiling. Ideal for epitope binning, fragment screening, and validating specificity in complex matrices.

2. Biolayer Interferometry (BLI)

BLI monitors binding via shifts in interference patterns of light reflected from a biosensor tip. Ligands are immobilized on the tip, and binding-induced changes in optical thickness are measured in real time.

High-throughput, low-sample-volume method compatible with crude samples (e.g., cell lysates). Requires minimal immobilization optimization.

3. Grating-Coupled Interferometry (GCI)

GCI measures phase shifts between a sample and reference beam in a fiber optic waveguide. Binding events alter the waveguide’s refractive index, enabling precise determination of K_D and kinetics.

Label-free, high-sensitivity analysis for low-abundance targets. Suitable for studying weak interactions (micromolar to millimolar K_D).

4. Isothermal Titration Calorimetry (ITC)

ITC quantifies heat exchange during binding. Incremental ligand injections into a protein solution release or absorb heat, revealing K_D, stoichiometry, enthalpy, and entropy.

Provides full thermodynamic profiles without labeling. Ideal for small-molecule/protein or protein/protein interactions.

5. Enzyme-Linked Immunosorbent Assay (ELISA)

Ligands are immobilized on a plate, and serially diluted protein samples are added. Bound proteins are detected using enzyme-conjugated antibodies and colorimetric substrates.

High-throughput, cost-effective screening for qualitative or semi-quantitative affinity ranking. Limited to endpoint measurements.

STEMart Provides:

Method Development & Validation: Tailored protocols for SPR, BLI, ITC, and GCI.
High-Throughput Screening: Rapid affinity ranking of drug candidates using FP or ELISA.
Biosimilar Comparability: Head-to-head binding studies against reference products.
Regulatory Support: ICH Q2(R1)/Q6B-compliant reports for IND/BLA submissions.
Advanced Kinetic Analysis: Global fitting of Konand Koff using the WAVEsystem or Kinetics Guide principles.

Ligand binding affinity evaluation is a cornerstone of biopharmaceutical development, bridging structural biology with therapeutic efficacy. By integrating advanced techniques, STEMart delivers actionable insights to optimize drug candidates, mitigate risks, and accelerate regulatory approval.

For more information on our Ligand Binding Affinity Evaluation service or to discuss your specific requirements, please contact us today.