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Qualitative and quantitative evaluation of S imon™, a new CE ‐based automated Western blot system as applied to vaccine development
Author(s) -
Rustandi Richard R.,
Loughney John W.,
Hamm Melissa,
Hamm Christopher,
Lancaster Catherine,
Mach Anna,
Ha Sha
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200095
Subject(s) - blot , capillary electrophoresis , isoelectric focusing , chromatography , chemistry , western blot , polyclonal antibodies , horseradish peroxidase , antibody , biology , biochemistry , enzyme , immunology , gene
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 S imple W estern™ (or S imon™) 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 S imon™ can quantitate specific components in one of our vaccine candidates and it provides good reproducibility and intermediate precision with CV <10%.