Epitope Binning Services by ForteBio Octet® Platform

When multiple antibodies have similar affinities for a target, determining whether they bind to the same or different epitopes on the target molecule becomes an inevitable step towards evaluating their individual functional potential. Using our Octet system, Creative biolabs can conduct the epitope binning analysis using samples either purified or directly from hybridoma supernatant or similar matrices.

Octet^® system performs sample analysis in 96- and 384-well format, processing up to 16 samples in parallel and requiring only 80 μL-120 μL/well samples in 384 well plates. Antibodies can be assayed in crude mixtures (cell lysates or hybridoma supernatants) or in DMSO (up to 10%) and glycerol, thus drastically reduce the need for sample preparation. The analytical capability of the platform ensures significantly easier, faster and better characterization of antibodies, helping researchers further characterize the specificity of the clones and their ability to block target activity, which can have far-reaching effects on the candidate's efficacy and pharmacokinetics.

Fig. 1 Octet data for an array of mAbs binding to Ag that was immobilized in different ways (as indicated in the plot titles). (Abdiche et al. 2012)

Other optional monoclonal antibody epitope binning services:

• Epitope Binning by Biacore Systems
• Epitope Binning by IBIS MX96 & CFM
• Epitope Binning by ProteOn Platform

Published Data

• Determination of the Binding Affinity of 2H2 to VP1 and MED25 by the ForteBio Octet System

Fig. 2 Location of the common epitope on the viral capsid surface. (Peihu Fan, 2015)

Enterovirus 71 (EV71) is the main pathogen of hand, foot, and mouth disease, but its pathogenesis in the central neuronal system is still unclear. To study the pathogenesis, the researchers identified a common epitope (co-epitope) between EV71 VP1 and human-mediated complex subunit 25 (MED25), which is highly expressed in the brainstem. They prepared monoclonal antibodies (2H2) against co-epitopes and identified their interaction with MED25 by assays in vitro and in vivo. In addition, through the ForteBio Octet system, the researchers determined that 2H2 can bind to VP1 and MED25. It was found that seven days after EV71 infection, 2H2 antibodies injected intravenously were distributed in the brainstem of mice. At the same time, they also detected 2H2-like antibodies in the sera of patients with the EV71 infection. These findings suggest that EV71 infection induces the production of antibodies and may further trigger an autoimmune response that leads to nervous system diseases.

FAQ

1. What is epitope binning, and how is it performed on the ForteBio Octet Platform?

   Epitope binning is a technique used to categorize monoclonal antibodies based on their binding to specific antigenic sites or epitopes on a target molecule. On the ForteBio Octet Platform, epitope binning is performed using a label-free, real-time detection system. Antibodies are immobilized on biosensor tips, and their interactions with antigens are monitored in the presence of competing antibodies. This method helps in identifying whether antibodies bind to the same, overlapping, or different epitopes.

2. Can the ForteBio Octet Platform handle high-throughput epitope binning?

   ForteBio Octet Platform is well-suited for high-throughput epitope binning. It can analyze multiple interactions simultaneously using a range of biosensors compatible with different types of antibodies and antigens. This capability allows for the rapid screening of a large panel of antibodies against multiple antigens, making it an efficient tool for large-scale antibody discovery and characterization projects.

3. What are the advantages of using the ForteBio Octet Platform for epitope binning compared to other methods?

   The ForteBio Octet Platform offers several advantages for epitope binning, including label-free detection, which preserves the native state of the antibodies and antigens without the need for fluorescent or radioactive labeling. It also provides real-time data, enabling kinetic and affinity measurements of antibody-antigen interactions. Additionally, its ability to perform assays in crude samples like cell supernatants or serum further enhances its utility in a wide range of settings, from research laboratories to biopharmaceutical companies.

4. What types of biosensors are used for epitope binning on the ForteBio Octet Platform?

   The ForteBio Octet Platform utilizes a variety of biosensor tips designed to accommodate different types of antibody-antigen interactions. Common biosensors include Protein A, Protein G, and anti-human Fc capture (AHC) sensors, which are used to immobilize antibodies through their Fc regions. Additionally, custom biosensors can be created by directly immobilizing specific antigens or antibodies, allowing for tailored assay conditions that match the unique requirements of each epitope binning study.

5. How does the ForteBio Octet Platform handle competition assays for epitope binning?

   In competition assays on the ForteBio Octet Platform, one antibody is immobilized on the sensor tip, and the antigen is introduced to allow binding. Subsequently, a second antibody is introduced to the mixture. If the second antibody fails to bind in the presence of the first, this suggests that both antibodies target the same epitope or overlapping epitopes. The real-time, kinetic monitoring capabilities of the Octet system provide detailed insight into the competitive dynamics of antibody binding.

6. What are the sample requirements for epitope binning using the ForteBio Octet Platform?

   The ForteBio Octet Platform is versatile in handling a wide range of sample types, including purified proteins, crude cell lysates, and culture supernatants. The minimal sample volume required for assays typically ranges from a few microliters to several hundred microliters, making it suitable for experiments where sample conservation is crucial. The system's robustness in different matrices ensures accurate epitope binning even in complex biological samples.

7. Can epitope binning on the ForteBio Octet Platform differentiate between conformational and linear epitopes?

   ForteBio Octet Platform is capable of differentiating between conformational and linear epitopes based on the nature of antibody-antigen interactions. By manipulating the conditions under which antibodies are tested—such as pH, ionic strength, and presence of detergents—the conformational state of proteins can be altered, influencing the binding behavior observed in the assays. This enables researchers to infer whether antibodies recognize epitopes that are conformation-dependent or linear sequences of amino acids.

Resources

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All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.