Residual bovine serum albumin (BSA) quantitation in vaccines using automated Capillary Western technology

Loughney, John W.; Lancaster, Catherine; Ha, Sha; Rustandi, Richard R.

doi:10.1016/j.ab.2014.05.004

Cited by 50 publications

(26 citation statements)

References 20 publications

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“…This further emphasises the value of using orthogonal techniques to quantify proteins across a wide dynamic range, or small proteins where options for Q-peptides are restricted. In our hands both quantitative platforms demonstrated an equivalently high level of precision using DOSCAT, which was within the range typically reported for SRM and QWB (using Simple Western technology) assays32728. Using ‘classic’ western blotting, it would be anticipated that reproducibility would be lower due to the greater number of manual handling steps.…”

Section: Resultssupporting

confidence: 80%

DOSCATs: Double standards for protein quantification

Bennett

Simpson

Holman

et al. 2017

Sci Rep

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The two most common techniques for absolute protein quantification are based on either mass spectrometry (MS) or on immunochemical techniques, such as western blotting (WB). Western blotting is most often used for protein identification or relative quantification, but can also be deployed for absolute quantification if appropriate calibration standards are used. MS based techniques offer superior data quality and reproducibility, but WB offers greater sensitivity and accessibility to most researchers. It would be advantageous to apply both techniques for orthogonal quantification, but workflows rarely overlap. We describe DOSCATs (DOuble Standard conCATamers), novel calibration standards based on QconCAT technology, to unite these platforms. DOSCATs combine a series of epitope sequences concatenated with tryptic peptides in a single artificial protein to create internal tryptic peptide standards for MS as well as an intact protein bearing multiple linear epitopes. A DOSCAT protein was designed and constructed to quantify five proteins of the NF-κB pathway. For three target proteins, protein fold change and absolute copy per cell values measured by MS and WB were in excellent agreement. This demonstrates that DOSCATs can be used as multiplexed, dual purpose standards, readily deployed in a single workflow, supporting seamless quantitative transition from MS to WB.

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Section: Resultssupporting

confidence: 80%

DOSCATs: Double standards for protein quantification

Bennett

Simpson

Holman

et al. 2017

Sci Rep

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“…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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“…BSA has the same composition as HSA [6,7]. So far, a lot of methods have been established for the detection and determination of BSA such as light scattering technique [10], near-infrared diffusion reflectance spectroscopy [11], capillary electrophoresis [12], molecular imprinted polymer (MIP) based electrochemistry [13] and flow injection chemiluminescence [14]. Although some of these methods display high selectivity and good sensitivity, they suffer from complicated sensor pretreatments and instrumentations, high costs and skilled operators requirements.…”

Section: Introductionmentioning

confidence: 99%