Abstract
Kinetic analysis of antibodies is one of the important studies for characterization of antibodies and screening of ligands. In our recent study (Ingale et al., 2014), we compared the antigenic profiles and binding characteristics of four HIV-1 envelope glycoprotein (Env) core immunogens using multiple monoclonal antibodies by Bio-Layer Light Interferometry (BLI). This technology enables real-time analysis of interactions on the surface of a fiber optic biosensor by accurately measuring kinetic constants such as Ka, Kd, and KD in a 96-well format.
Materials and Reagents
Equipment
Software
Procedure
Biosensors are tips coated with proprietary biocompatible matrix that can bind to Fc region of human immunoglobulin with minimal non-specific binding. In a separate black bottom plate, the biosensors were hydrated in 200 μl PBS for 10 min at room temperature prior to the experiment. The sample plate was generated according to the template shown in Figure 1. The buffer wells were filled with 200 μl PBS. Selected monoclonal antibodies were diluted to 5 μg/ml in PBS and dispensed at a volume of 200 μl into each well of the loading column as indicated in the template. The H1 well was filled with 200 μl PBS. The analyte (HIV envelope core glycoprotein is used in the example shown in Figure 1) was 2-fold serially diluted in PBS starting from 1 μM to 31.25 nM in a separate plate and transferred in a 200 μl volume in the “association column” of the template as shown (Figure 1). The sensor plate (plate with bio-sensors) and sample plate (plate with other reagents, as shown in Figure 1) were then inserted into the Octet Red machine and a basic kinetic measurement experiment was performed as follows:
Acknowledgments
This protocol was previously used in Ingale et al. (2014).
References
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