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Two-Dimensional Electrophoresis Coupled with Western Blot as a Method to Detect Potential Neutralizing Antibody
Milan Obradovic1, Heather L Wilson2
1Faculté de médecine vétérinaire/Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, QC, Canada.
Antigen selection is a critical step in subunit vaccine design, especially if the goal is to identify antigens that can be bound by neutralizing antibodies to prevent invasion of cells by intracellular bacteria. Here, we describe a method involving two dimensional gel electrophoresis (2-DE) coupled with western blotting (WB) and mass spectrometry (MS) to identify bacterial proteins that: (1) interact with the host target cell proteins, and (2) are targeted by antibodies from sera from infected animals. Subsequent steps would be performed to validate that the bacteria are targeted by neutralizing antibodies to prevent invasion of the eukaryotic cells.
Antigen selection is a critical step in subunit vaccine design, especially if the goal is to identify antigens that can be bound by neutralizing antibodies to prevent invasion of cells by intracellular bacteria. Here, we describe a method involving two dimensional gel electrophoresis (2-DE) coupled with western blotting (WB) and mass spectrometry (MS) to identify bacterial proteins that: (1) interact with the host target cell proteins, and (2) are targeted by antibodies from sera from infected animals. Subsequent steps would be performed to validate that the bacteria are targeted by neutralizing antibodies to prevent invasion of the eukaryotic cells.
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