Antibody affinity measurement is critical for understanding how tightly an antibody binds to its target antigen, which is key to determining its effectiveness in biological assays, therapeutic applications, and vaccine development. Several techniques can be used to measure the affinity of antibodies for their antigens, with each method offering different advantages depending on the type of experiment, the nature of the antibody, and the binding conditions.
Key Terminology
Affinity: The strength of the interaction between a single antigen-binding site of an antibody and its corresponding epitope on an antigen.
KD (Dissociation Constant): A quantitative measure of antibody affinity. Lower KD values indicate higher affinity (stronger binding), while higher KD values indicate lower affinity (weaker binding).
Techniques for Measuring Antibody Affinity
Surface Plasmon Resonance (SPR)
Principle: SPR measures real-time binding interactions between an antibody and an antigen immobilized on a sensor chip. As the antibody binds to the antigen, changes in the refractive index on the chip surface are detected, providing information on both the association (on-rate) and dissociation (off-rate) of the antibody-antigen interaction.
KD Calculation: The KD value is calculated as the ratio of the dissociation rate constant (k_off) to the association rate constant (k_on):
KD = Koff \ Kon
Advantages
Provides both kinetic and equilibrium affinity data.
Can measure interactions in real-time without labeling.
Suitable for a wide range of affinities, including very low KD values.
Disadvantages
Requires specialized equipment (e.g., Biacore).
Immobilization of antigens may affect the interaction depending on orientation and density.
Applications
Frequently used in drug development, therapeutic antibody screening, and protein-protein interaction studies.
Biolayer Interferometry (BLI)
Principle: Similar to SPR, BLI measures the interference pattern of light reflected from the surface of a sensor where an antigen is immobilized. When an antibody binds to the antigen, the interference pattern shifts, and both association and dissociation rates can be determined.
KD Calculation: Like SPR, KD is calculated as the ratio of the dissociation rate (k_off) to the association rate (k_on).
Advantages
Label-free, real-time measurement.
Does not require extensive sample preparation.
Works well in a wide variety of buffers and experimental conditions.
Disadvantages
Immobilization on the sensor surface can affect binding properties.
Applications
Used for antibody affinity measurement in drug discovery, protein engineering, and structural biology.
Enzyme-Linked Immunosorbent Assay (ELISA) for Affinity
Principle: ELISA can be adapted to estimate antibody affinity by measuring the interaction between an antigen and antibody under equilibrium conditions. A high-affinity antibody will remain bound to its antigen even at low concentrations of antigen, while low-affinity antibodies will dissociate more easily.
KD Estimation:
One common approach is to coat an antigen on an ELISA plate, incubate with various concentrations of the antibody, and then detect bound antibodies with a labeled secondary antibody.
A Scatchard plot or nonlinear regression analysis can be used to estimate the KD from the ELISA binding curves.
Advantages
Simple and widely available method.
Does not require specialized equipment.
Disadvantages
Does not provide real-time kinetic data (association/dissociation rates).
The affinity measurements are approximate, and this method is less sensitive for very high-affinity antibodies.
Applications
Used for routine affinity estimation in antibody development, including hybridoma screening.
Equilibrium Dialysis
Principle: Equilibrium dialysis is a classic method where an antibody and its antigen are separated by a semipermeable membrane that allows free diffusion of the antigen. The system is allowed to reach equilibrium, and the concentration of free antigen and antigen-bound antibody is measured on both sides of the membrane.
KD Calculation: The KD is determined from the ratio of bound to free antigen at equilibrium.
Advantages
Provides an accurate measure of KD, particularly for small antigens such as peptides or small molecules.
Disadvantages
Time-consuming and requires significant sample material.
Not suitable for real-time measurement or large antigens.
Applications
Primarily used for small molecules and peptide-antibody interactions.
Isothermal Titration Calorimetry (ITC)
Principle: ITC measures the heat released or absorbed during the interaction between an antibody and an antigen. The heat change is directly proportional to the amount of binding, allowing for the calculation of the binding affinity.
KD Calculation: The ITC experiment provides a direct measure of the binding constant (KD), as well as the stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) of the interaction.
Advantages
Label-free and provides thermodynamic information (ΔH, ΔS).
Can measure very weak to strong binding affinities.
Disadvantages
Requires a large amount of sample (milligram quantities).
Less suitable for very high-affinity interactions (low KD values).
Applications
Widely used in biophysics to study antibody-antigen interactions and in the development of monoclonal antibodies.
Fluorescence Polarization (FP)
Principle: Fluorescence polarization measures the binding affinity between a fluorescently labeled antigen and an antibody. When the fluorescent antigen is free in solution, it rotates quickly, resulting in low polarization of emitted light. When the antigen binds to an antibody, the rotation slows, leading to higher polarization.
KD Calculation: The KD is determined by measuring the polarization of light as a function of increasing antibody concentration.
Advantages
Fast and suitable for high-throughput screening.
Works well for small molecules and peptides.
Disadvantages
Requires labeling of the antigen, which may affect binding.
Limited to interactions with small or medium-sized antigens.
Applications
Often used for small molecule-protein interactions, peptide-antibody interactions, and screening assays.
Flow Cytometry for Affinity Measurement (FACS-Based Binding Assays)
Principle: Flow cytometry can be adapted to measure the binding of antibodies to cell-surface antigens by incubating cells with varying concentrations of fluorescently labeled antibodies. The mean fluorescence intensity (MFI) is proportional to the binding of the antibody to the antigen.
KD Calculation: By plotting MFI as a function of antibody concentration, the KD can be estimated using nonlinear regression analysis.
Advantages
Allows measurement of antibody binding in the context of live cells and membrane-bound antigens.
Provides single-cell resolution and can be used for different cell populations simultaneously.
Disadvantages
Limited to membrane-bound antigens or antigens expressed on the surface of cells.
Applications
Used in immunology for measuring antibody affinities to cell-surface receptors or other membrane-associated antigens.
Comparison of Techniques for Antibody Affinity Measurement
| Method | KD Range | Kinetics | Sample Size | Real-Time | Labeling | Applications |
| SPR (Surface Plasmon Resonance) | pM–mM | Yes | Low | Yes | No | Kinetic analysis, therapeutic antibody screening |
| BLI (Biolayer Interferometry) | pM–mM | Yes | Low | Yes | No | Drug discovery, protein interaction studies |
| ELISA (for affinity) | nM–μM | No | Low | No | No | Routine affinity screening |
| Equilibrium Dialysis | pM–mM | No | High | No | No | Peptide-antibody interactions |
| Isothermal Titration Calorimetry (ITC) | nM–mM | No | High | No | No | Thermodynamic studies, antibody development |
| Fluorescence Polarization | nM–μM | No | Low | No | Yes | Small molecule-protein interactions |
| Flow Cytometry (FACS-based) | nM–μM | No | Medium | No | Yes | Antibody binding to cell-surface antigens |
Conclusion
The choice of technique for measuring antibody affinity depends on the nature of the interaction, the type of antigen (soluble or membrane-bound), and the resources available. SPR and BLI are popular for real-time kinetic studies, while ELISA and ITC are commonly used for equilibrium affinity measurements. Each method has its strengths and limitations, so researchers often choose the technique that best fits their experimental design and desired information.