Antibody Affinity Assays
What is Antibody Affinity?
Antibody affinity is a measure of the binding strength between an antibody and the epitope or antigenic determinant cluster of the antigen. It reflects how tightly the antibody binds to its antigen and is a result of non-covalent interaction forces, such as attraction between amino acids, hydrogen bonds, and hydrophobic interactions. Antibody affinity indicates the ability of an antibody molecule to interact with a half-antigen molecule or antigen molecule of a determinant cluster.
The magnitude of antibody affinity can be expressed by the affinity constant KD, which is calculated using the formula KD=[Ab▪H]/([Ab][H]) in liters per mole concentration. Here, [ ] denotes molar concentration, Ab denotes antibody, H denotes half-antigen, and Ab-H represents the affinity constant of the antibody-antigen complex. Higher KD values indicate a stronger ability of the antibody to bind to the half-antigen. The strength of antibody affinity depends on how well the paratope of the antibody and the epitope of the antigen coordinate, including factors such as the size of the contact area, degree of fit, and distribution of charged groups and hydrophobic groups.
Antibody Affinity Assay Technology Platform at Creative Proteomics
Biofilm Interferometry (BLI)
BLI analysis using the Fortebio Octet® detection instrument. Antibody affinity detection is achieved by real-time detection of changes in optical interference signals.
| Sample Requirements | Service Content | Analytical Projects | Delivery Content |
Protein, antibody (>150ug, purity >85%) Liquid samples require buffer for PBS, no glycerol added |
- Affinity screening: primary affinity screening of the antibodies to be tested, quickly finding the 4-5 groups of antibodies with the best affinity from a dozen groups of samples - Affinity determination: accurate determination of the affinity between antigen and antibody (accurate dissociation constant Kd, binding constant Ka, and affinity constant KD are measured) |
- Antibody-antigen affinity assay - Antibody affinity assay with FcγRs/FcRn/C1q - Protein-protein affinity assay - Protein and small molecule affinity assay - Supernatant Affinity Sorting - Epitope Binning |
Affinity determination report (including dissociation constant Kd, binding constant Ka, affinity constant KD) Other related reports |
BioLayer Interferometry (BLI) technology assay principle (Concepcion et al., 2009)
Surface Plasmon Resonance (SPR) Technology
Compared to competitive ELISA, SPR method has many advantages, such as real-time monitoring of the dynamic process of the reaction and better reproducibility of the experimental results.
Creative Proteomics has two systems, the Biacare 8K and Biacore T200, to provide quality SPR services. Not only can antibody affinity be measured but also binding kinetics, quantification, specificity, binding activity, stability, and specificity can be analyzed.
| Sample Requirements | Service Content | Analytical Projects | Delivery Content |
Protein, antibody (Sample concentration >1mg/mL, purity >90%) Analytes must not contain high refractive index substances such as glycerol, sucrose, imidazole, etc. Ligand does not contain Tris and other components containing primary amine groups |
Method development, optimization and affinity determination | - Optimization of ligands - Analyte concentration interval optimization, binding and dissociation time optimization |
Affinity determination report (including dissociation constant Kd, binding constant Ka, affinity constant KD) Other related reports |
General scheme of the SPR-based assay for simultaneous determination of IFX and ATI concentrations in serum (Beeg et al., 2019).
Why is it necessary to analyze antibody affinity?
Once recombinant antibodies are produced, their affinity must be determined to ensure successful downstream applications. In immunological experiments such as RIA, ELISA, and IFA, the affinity of the antibody to the specific target antigen must be above a certain threshold to ensure the sensitivity and reliability of the measurement. When using an antibody conjugated to a solid phase for affinity purification of the antigen, an antibody of moderate affinity is needed to avoid strong elution conditions that could cause antigen denaturation, allowing for mild elution conditions.
Since general polyclonal antibody sera are mixtures of antibodies with vastly different affinities, different components of corresponding affinities can be used for various purposes. However, when replacing polyclonal antibody sera with monoclonal antibodies, affinity must be determined to select suitable backups. Furthermore, since the specific affinity of some monoclonal antibodies for a particular antigen can vary greatly with changes in environmental conditions such as temperature, pH, and ion strength, even slight changes, when selecting monoclonal antibodies for a particular purpose, it is necessary to measure their affinity under conditions similar to those in actual applications.
References
- Concepcion, Joy, et al. "Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization." Combinatorial chemistry & high throughput screening 12.8 (2009): 791-800.
- Beeg, Marten, et al. "A surface plasmon resonance-based assay to measure serum concentrations of therapeutic antibodies and anti-drug antibodies." Scientific Reports 9.1 (2019): 2064.