Platforms and Techniques Used for Biomarker Assays: Where are We Now?

Hottenstein, Charles; Szapacs, Matthew; Fuller, Kerensa J.; Evans, Christopher A.

doi:10.4155/bio-2017-0107

Cited by 7 publications

(2 citation statements)

References 10 publications

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“…Biofluid cytokine concentrations are a particularly widely measured, diverse group of proteins used to estimate immune activation processes in various healthy and diseased populations; a tool that now spans across disciplines (e.g., psychiatry, neurology) and in large multisite clinical studies 1 . Advancements in technologies have facilitated ease and pragmatics of biofluid marker quantification by allowing for simultaneous analysis of multiple markers via multiplexing, higher analytical sensitivity and lower limits of detection and quantification, and reduction in the amount of sample needed (e.g., compared to standard ELISA) 2–4 . However, the growing number of available platforms with slightly differing technologies measuring the same protein markers has made it difficult to not only select the most optimal quantification tool, but to compare marker results across platforms and studies.…”

Section: Introductionmentioning

confidence: 99%

A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?

et al. 2018

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HPE and Simoa showed comparable detectability and intra-assay precision measuring a panel of commonly examined cytokine proteins, with the exception of IL-1β which was not reliably detected on HPE. However, Simoa demonstrated overall higher concentrations and the two platforms did not show agreement when directly compared against one another. Relative cytokine ratios and associations demonstrated similar patterns across platforms. Absolute cytokine concentrations may not be directly comparable across platforms, may be analyte dependent, and interpretation may be best limited to discussion of relative associations.

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Section: Introductionmentioning

confidence: 99%

A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?

et al. 2018

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“…Matrix simplification and target enrichment can be accomplished by enriching either the target protein (protein level) or target signature peptide (SP) after digestion (peptide level), mostly using immunoaffinity enrichment approaches. , Among these methods, peptide-level enrichment has several unique advantages. For instance, biases and variations of the enrichment process can be readily corrected by spiking in stable-isotope-labeled-peptide internal standards (SIL-peptide-IS) before enrichment, which are quite cost-effective to produce and readily available; moreover, because the digested samples are usually cleaned up prior to enrichment, the process is not affected by detrimental compounds such as detergents, lipids, and others, and thereby works well in tissue samples. , A number of works have demonstrated greatly improved quantitative sensitivity via peptide-level enrichment. − Nonetheless, the development of an effective antipeptide antibody is usually quite challenging, time-consuming, and costly, which is often not practical at the early drug-discovery and -development stage. , …”

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confidence: 99%

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths

Zhang

et al. 2019

Anal. Chem.

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Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption–desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI – 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87–95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.

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An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

An¹,

Sikorsiki²,

Kellie³

et al. 2022

Journal of Chromatography A

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Platforms and Techniques Used for Biomarker Assays: Where are We Now?

Cited by 7 publications

References 10 publications

A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?

A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths

An antibody-free platform for multiplexed, sensitive quantification of protein biomarkers in complex biomatrices

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