Qualitative Kinetic Assays

Real-time monitoring of biomolecular interactions provides more immediate information in situations where end-point readouts require time to develop a single number. ForteBio’s Octet family of instruments is effective in answering questions that traditional ELISA type assays can’t.

While screening clones to find a target’s best binder, you may find that multiple candidates all have approximately the same affinity. More information about the epitopes of the targets can help identify new epitopes on a target protein or eliminate binders that don’t block target activity. With the Octet System or Octet RED System, you can quickly and easily determine if multiple clones share the same epitope or recognize different regions of the target protein.

As shown in Figure 1, antibodies 1,2, and 3 all recognize a different epitope than antibody A. Since antibody 4 did not bind when exposed to the antibody A/target complex, appears that antibody A and antibody 4 share the same epitope on the target.

Detecting different antibody isotypes in the presence of background proteins is quick and easy with BLI-based technology. The Octet family of instruments provides a simple method for rapidly identifying both heavy and light chain subclasses.

A biosensor immobilized with an anti-species antibody will capture that particular antibody from a crude mixture, such as a hybridoma sup. Exposure to a series of isotype-specific antibodies provides a sandwich format for quickly identifying the antibody’s isotype out of a crude mixture. The Octet System and Octet RED System provide simultaneous monitoring of eight biosensors, so you can screen for up to eight different isotypes in a sample — in about three minutes, with no assay development or complicated wash steps.

Screening for potential binding pairs using an Octet System takes a fraction of the time it would take through traditional methods. Optimal binding pair selection can greatly speed up the time needed to create new binding assays such as an ELISA. By immobilizing a single ligand onto the eight biosensors, the binding of any analytes loaded into the 96-well sample plate can be quickly detected. Once the assay is complete, a qualitative assessment of the results will identify which analytes bound best to the immobilized ligand.

Figure 3 illustrates the real-time binding chart of the antibody pair screen. Two pairs can be quickly identified as suitable for further assay development. Ab1 capture antibody with detection antibody Ab5 exhibits good binding to the analyte. The interaction between Ab1/Ab5 also exhibits some dissociation, indicating a weaker affinity. Ab2 capture antibody with Ab4 detection antibody exhibits both good binding and affinity.