Qualitative and quantitative evaluation of <scp>S</scp>imon™, a new <scp>CE</scp>‐based automated Western blot system as applied to vaccine development

Rustandi, Richard R.; Loughney, John W.; Hamm, Melissa; Hamm, Christopher; Lancaster, Catherine; Mach, Anna; Ha, Sha

doi:10.1002/elps.201200095

Cited by 55 publications

(49 citation statements)

References 22 publications

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“…Once the proteins were separated within the pH gradient they were exposed to UV light and covalently cross-linked to a light-activated volume-accessible gel present in the microchip. This technique gave similar capture efficiencies (≈ 0.01%) to previous reports where proteins were immobilized on the inner surface of the capillary [42, 143]. Wash steps were performed by electrophoretic transport on the immobilized protein without concern of sample loss.…”

Section: Microchip Electrophoresissupporting

confidence: 55%

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Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

2014

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The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis.

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Section: Microchip Electrophoresissupporting

confidence: 55%

“…Simon™ also makes quantitative Western blots possible. Using this instrument, a standard curve was generated for a vaccine candidate protein with linearity from 0.45–7 µg/mL and R 2 values of 0.990 or greater for five experiments [42]. …”

Section: Techniquesmentioning

confidence: 99%

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

2014

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“…However, several of the concerns that plague non-automated western blotting will also affect automated western blotting. The use of capillary columns in some new automated machines [62,63] introduces new technical concerns including how we can relate some of the results from capillary electrophoresis westerns back to traditional western blotting results.…”

Section: Other Problems Associated With Inaccurate Quantification Of mentioning

confidence: 99%

The necessity of and strategies for improving confidence in the accuracy of western blots

Ghosh¹,

Gilda²,

Gomes³

2014

Expert Review of Proteomics

235

178

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Summary Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts, however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

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“…Rustandi and colleagues applied size-CNIA to protein expression and identity in vaccine development. They reported that the technology increased the efficiency of analytical testing and could more effectively guide process development [63]. The group also developed a new improved method for detection of residual BSA to meet quality control requirements in vaccine production [64].…”

Section: Applicationsmentioning

confidence: 99%

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics

2015

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There is an emerging demand for the use of molecular profiling to facilitate biomarker identification and development, and to stratify patients for more efficient treatment decisions with reduced adverse effects. In the past decade, great strides have been made to advance genomic, transcriptomic and proteomic approaches to address these demands. While there has been much progress with these large scale approaches, profiling at the protein level still faces challenges due to limitations in clinical sample size, poor reproducibility, unreliable quantitation, and lack of assay robustness. A novel automated capillary nano-immunoassay (CNIA) technology has been developed. This technology offers precise and accurate measurement of proteins and their post-translational modifications using either charge-based or size-based separation formats. The system not only uses ultralow nanogram levels of protein but also allows multi-analyte analysis using a parallel single-analyte format for increased sensitivity and specificity. The high sensitivity and excellent reproducibility of this technology make it particularly powerful for analysis of clinical samples. Furthermore, the system can distinguish and detect specific protein post-translational modifications that conventional Western blot and other immunoassays cannot easily capture. This review will summarize and evaluate the latest progress to optimize the CNIA system for comprehensive, quantitative protein and signaling event characterization. It will also discuss how the technology has been successfully applied in both discovery research and clinical studies, for signaling pathway dissection, proteomic biomarker assessment, targeted treatment evaluation and quantitative proteomic analysis. Lastly, a comparison of this novel system with other conventional immuno-assay platforms is performed.

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Qualitative and quantitative evaluation of Simon™, a new CE‐based automated Western blot system as applied to vaccine development

Cited by 55 publications

References 22 publications

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

The necessity of and strategies for improving confidence in the accuracy of western blots

Capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics

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