Kinetic Characterization of Antibody/Antigen Interactions

A leading application of biomolecular interaction analysis technology is the characterization of antibodyantigen interactions. By measuring the association and dissociation rates of the interaction, antibodies with desired binding characteristics can be identified. A rapid association rate and a slow dissociation rate may be indicative of therapeutic efficacy. Affinity maturation programs exist to develop antibodies with improved binding affinity, which can be quantitatively ranked using biomolecular interaction analysis technology. Antibodies are useful diagnostic reagents, and are frequently utilized as tandem pairs in a sandwich assay format. In a common sandwich assay format, one antibody serves to capture analyte, and the second antibody, binding to a different epitope on the analyte, serves to directly or indirectly carry the detection label. One advantage to sandwich assays is increased sensitivity via signal amplification by the second binding antibody. While a sandwich assay format may be desirable, a second antibody partner is not always available. A single ligand assay design can be utilized in cases where a sandwich assay design is not feasible. In a single ligand assay, one antibody captures analyte, and detection can occur by labeling the analyte, or by using label-free detection technologies.[1] Conventional biomolecular interaction analysis technologies such as surface plasmon resonance (SPR) can enable a single ligand assay format, but may not deliver adequate analyte detection sensitivity. Antibodies immobilized in the 100–200 nm deep hydrogel biosurface are typically immobilized as a monolayer, limiting the analyte capture capacity of this technology. Nanopore optical interferometry (NPOI), on the other hand, utilizes a 1.5–2.0 μm deep nanoporous silicon biosurface, increasing the surface area available for interaction analysis by 100 fold over SPR. Ligands are immobilized in multiple layers in the nanoporous silicon biosurface, increasing the analyte capture capacity and the sensitivity of this technology. NPOI is used to measure antibody-antigen interactions in real time in a label-free format. In addition to determining antigen concentration, SKi Pro can characterize the kinetics of the antibody-antigen binding interaction, quantitating the on rate (kon), off rate (koff ) and affinity (KD) of the antibody to its antigen. This application note demonstrates the use of SKi Pro to characterize the binding of whole molecule antibodies to their protein antigens. Antigens of molecular weights from 6–83 kilodaltons were analyzed. The sensitivity of this methodology is further illustrated by measuring the limit of detection (LOD) of the antibodies to concentrations of the smallest of these analytes.