The Octet system uses Bio-Layer Interferometry (BLI), an optical analytical technique that allows for label-free, real-time monitoring of bi molecular interactions. As the biosensor tip is exposed to different analytes the number of molecules bound to the biosensor tip will change. This causes a change in the optical layer thickness and results in a shift in the interference pattern that can be measured in real time.
Our murine antibodies (GMA-092 and GMA-095) show specific binding to human prothrombin on ELISA. We used the Octet to determine if they bind the same epitope on prothrombin. The results below are with sensors loaded with GMA-095 and show that GMA-092 binds a distinct epitope from GMA-095:
Each new step in the method is denoted by a vertical dashed red line (9 steps total):
Step 1: Baseline – kinetics buffer to establish a baseline for the next step.
Step 2: Capture antibody (GMA-095) binds to the sensor.
Step 3: Baseline.
Step 4: Analyte of interest (prothrombin) binds to the capture antibody.
Step 5: Baseline. This step can also be used to access the dissociation curve of the analyte from the capture antibody.
Step 6: Quench. This step is used to fill any empty antibody binding sites on the sensor tip so that all subsequent binding should be only to the analyte of interest. In this example Ms IgG is used.
Step 7: Baseline.
Step 8: Binding of second antibody to the analyte of interest (prothrombin). The blue line represents GMA-092 and shows binding to prothrombin. The red line represents additional GMA-095 and shows the expected lack of additional binding, consistent with its prothrombin binding site already being occupied.
Step 9: Dissociation. The stability of the sandwich protein complex is monitored in kinetics buffer. Minimal dissociation is seen here.