title: Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development.
abstract: Many CE-based technologies such as imaged capillary IEF, CE-SDS, CZE, and MEKC are well established for analyzing proteins, viruses, or other biomolecules such as polysaccharides. For example, imaged capillary isoelectric focusing (charge-based protein separation) and CE-SDS (size-based protein separation) are standard replacement methods in biopharmaceutical industries for tedious and labor intensive IEF and SDS-PAGE methods, respectively. Another important analytical tool for protein characterization is a Western blot, where after size-based separation in SDS-PAGE the proteins are transferred to a membrane and blotted with specific monoclonal or polyclonal antibodies. Western blotting analysis is applied in many areas such as biomarker research, therapeutic target identification, and vaccine development. Currently, the procedure is very manual, laborious, and time consuming. Here, we evaluate a new technology called Simple Western™ (or Simon™) for performing automated Western analysis. This new technology is based on CE-SDS where the separated proteins are attached to the wall of capillary by a proprietary photo activated chemical crosslink. Subsequent blotting is done automatically by incubating and washing the capillary with primary and secondary antibodies conjugated with horseradish peroxidase and detected with chemiluminescence. Typically, Western blots are not quantitative, hence we also evaluated the quantitative aspect of this new technology. We demonstrate that Simon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.
authors.affiliation: Vaccine Analytical Development, Merck Research Laboratories, West Point, PA, USA. richard_rustandi@merck.com
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.forename: John W
authors.initials: JW
authors.lastname: Loughney
authors.forename: Melissa
authors.initials: M
authors.lastname: Hamm
authors.forename: Christopher
authors.initials: C
authors.lastname: Hamm
authors.forename: Catherine
authors.initials: C
authors.lastname: Lancaster
authors.forename: Anna
authors.initials: A
authors.lastname: Mach
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
mesh_terms: D015153:Blotting, Western / Q000295:instrumentation* / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D012371:Robotics / Q000295:instrumentation; D012680:Sensitivity and Specificity; D014612:Vaccines / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D011506:Proteins; D014612:Vaccines