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

Abstract

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.

P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy

“…This is lower than the published variation coefficient (10%) for size-based WES 59 and indicates that peaks for P-gp and β -actin are reproducible and can be compared between lanes. These data also demonstrate that the WES assay allows us to detect and quantify P-gp and β -actin simultaneously in human brain capillary samples at the nanogram level.…”

Multiplexed MRM‐based assays for the quantitation of proteins in mouse plasma and heart tissue

“…The implementation of stable isotope‐labeled standard (SIS) peptides in MRM assays, for example, has been thus far demonstrated for the quantitation of small protein panels in various mouse biosamples . Absolute protein quantitation in mouse biosamples has also been achieved with immunoassays . Each technique has its advantages (e.g., sensitivity, specificity, robustness), and its disadvantages (e.g., development time, cross‐reactivity, availability, transferability) that must be considered before development and implementation of an assay.…”

Recent advances in CE and microchip‐CE in clinical applications: 2014 to mid‐2017

“…The development of immunoassays in a CE or ME format has become a popular analytical technique although only a few reports have been applied to clinical analysis. In either format, antibodies are either incorporated in an immunoaffinity capture device as a preanalysis selector prior to CE separation and detection of the captured analytes or the immunoassay is performed in the capillary itself and the end‐products analyzed by CE . Guzman and Guzman described an on‐line immune‐extraction system or immune‐concentrator that is able to capture analytes of interest in biological fluids prior to analysis by CE.…”