Protein Quantification in Complex Mixtures by Solid Phase Single-Molecule Counting

Tessler, Lee A.; Reifenberger, Jeffrey G.; Mitra, Robi D.

doi:10.1021/ac901068x

Cited by 63 publications

(60 citation statements)

References 37 publications

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“…The available technologies for sensitive protein detection typically use pairs of affinity reagents in a sandwich configuration. This is true for the popular sandwich ELISA (40), and for more recent highly sensitive techniques such as the biobarcode assay (41), singlemolecule counting assays (42)(43)(44), and homogeneous PLA (36,37,45).…”

Section: Discussionmentioning

confidence: 98%

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Tavoosidana

Ronquist

Darmanis

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

201

173

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Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

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Section: Discussionmentioning

confidence: 98%

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Tavoosidana

Ronquist

Darmanis

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

201

173

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“…The sample preparation for this samples are already described BSA has been widely used as a blocking agent to limit non-specific adsorption of proteins [149][150][151][152][153]. In this section, the protocol for passivating the ES capillaries with BSA is described at pH conditions close to its isoelectric point (pI) of 4.8 [156].…”

Section: Methodsmentioning

confidence: 99%

“…To understand the effect of adsorbed proteins that desorb, the following approach For surface passivation BSA [149][150][151][152][153] and gelatin [154,155] were used as blocking agents, each of which can form monolayers or multiple layers on a surface and hence reduce surface adsorption of other proteins. …”

Section: Introductionmentioning

confidence: 99%

Electrospray–differential mobility analysis of bionanoparticles

Guha

Tarlov

et al. 2012

Trends in Biotechnology

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The growth of the multibillion dollar bionanoparticle industry has spurred the development of new physical characterization methods. One such method, electrospraydifferential mobility analysis (ES-DMA) constitutes an electrospray for aerosolization of bionanoparticles (such as viruses, gold-nanoparticles, proteins, nanoparticle-protein complexes) and an ion mobility method that operates at atmospheric conditions, and separates bionanoparticles spatially. This dissertation identifies some relevant "problem" areas for ES-DMA by reviewing selected applications.Some such problems are: proteins while passing through ES capillaries are found to interact with it and thus produce time dependent size distributions. Further, it is thought that adsorbed proteins may subsequently desorb and influence size distributions with the ES-DMA which may concomitantly affect quantification of aggregates. These artifacts are studied systematically and it is demonstrated that ES-DMA can quantify adsorption-desorption of complex protein mixtures at high shear rates. Further, it is shown that desorbing proteins do not have a significant effect on size distributions. Another artifact of the ES takes place during the aersolization process. Two units (called monomers) of a bionanoparticle may get encapsulated in the same ES droplet and upon drying of the droplet create artificial dimers thus affecting quantification with ES-DMA. Assuming Poisson distribution, this thesis provides a systematic approach that can be undertaken to eliminate this artifact. A third artifact arises from the low sensitivity of the DMA to size increase. When a ligand (for e.g. protein) adsorbs to a bionanoparticle it creates an increase in the size of the later, which can be used to quantify the amount of ligand adsorbed per bionanoparticle. As ligands can change conformations upon adsorption, using ES-DMA for such applications may be flawed. This issue has been identified and a solution has been provided by integrating a mass analyzer after the ES-DMA.After correcting for these artifacts, this dissertation delves into characterization of different types of bionanoparticles and demonstrates that ES-DMA has several advantages over other traditional techniques such as transmission electron microscopy, size exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and plaque assay and thus has immense potential to become a process analytical technique in biomanufacturing environments. ELECTROSPRAY-DIFFERENTIAL MOBILITY ANALYSIS OF BIONANOPARTICLES

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“…Single-molecule (SM) fluorescence microscopy studies hold great promise for elucidating biological systems [1], but the non-specific surface adsorption of fluorescently labelled proteins [2,3], antibodies [4] and bioconjugated nanoparticles [5] is often a significant source of experimental noise. Recently, low-background surface coatings have been developed that reduce protein adsorption to SM levels-levels at which a digital signal from individual target molecules can be reliably quantified above the background of non-specifically adsorbed molecules.…”

Section: Introductionmentioning

confidence: 99%

Nanogel surface coatings for improved single-molecule imaging substrates

Tessler

Donahoe

Garcia

et al. 2011

J. R. Soc. Interface.

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Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings. We characterized the nanogel coating to determine its suitability for SM protein experiments. SM adsorption counting revealed that nanogel-coated surfaces exhibit lower protein adsorption than covalently coupled BSA surfaces and monolayers of multi-arm PEG, so this surface displays one of the lowest degrees of protein adsorption yet observed. Additionally, the nanogel coating was resistant to DNA adsorption, underscoring the utility of the coating across a variety of SM experiments. The nanogel coating was found to be compatible with surfactants, whereas the BSA coating was not. Finally, applying the coating to a real-world study, we found that single ligand molecules could be tethered to this surface and detected with high sensitivity and specificity by a digital immunoassay. These results suggest that PEG -BSA nanogel coatings will be highly useful for the SM analysis of proteins.

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Protein Quantification in Complex Mixtures by Solid Phase Single-Molecule Counting

Cited by 63 publications

References 37 publications

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Electrospray–differential mobility analysis of bionanoparticles

Nanogel surface coatings for improved single-molecule imaging substrates

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