Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

Winters‐Hilt, Stephen; Morales, Eric; Amin, Iftekhar; Stoyanov, Alexander V.

doi:10.1186/1471-2105-8-s7-s20

Cited by 10 publications

(24 citation statements)

References 33 publications

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“…A preliminary description of antibody blockade studies on the nanopore detector, for a well defined synthetic polypeptide antigen, are given in [9,32]. Similar results are found IgG subclass 1 monoclonal antibodies for biotin, HIV, and anti-GFP, and all have produced similar signals.…”

Section: S3 Managing Common Interference Agents and Antibodies As Easupporting

confidence: 53%

“…For further details on aptamers in the context of nanopore transduction detection see [17][18][19], and in general see [20][21][22][23][24]. Further details on antibodies pertinent to nanopore detection are placed in the Suppl., including diagrammatic figures and structural details from various references [25][26][27][28][29][30][31][32].…”

Section: Managing Common Interference Agents and Aptamers And Antibomentioning

confidence: 99%

“…Further details on antibodies pertinent to nanopore detection, including diagrammatic figures (Suppl. Fig.s 5-7), and structural details from various references [25][26][27][28][29][30][31][32] are placed in the Suppl.…”

Section: Antibodies As Easily Identifiable Interference or Transducermentioning

confidence: 99%

“…The channel may provide a means to directly measure and characterize antibody Fc glycosylations, a critical quality control needed in antibody therapeutics to have correct human-type glycosylation profiles in order to not (prematurely) evoke an immunogenic response. [9,32], hydrophobic bonds are very difficult to characterize by existing crystallographic and other means, and often contribute half of the overall binding strength of the antigen-antibody bond. Hydrophobic groups of the biomolecules exclude water while forming lock and key complementary shapes.…”

Section: S1 Biotin-streptavidin Binding Studies Via Nanopore Event Tmentioning

confidence: 99%

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Biological system analysis using a nanopore transduction detector: from miRNA validation, to viral monitoring, to gene circuit feedback studies

Winters‐Hilt¹

2017

asms

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The system biologist is currently lacking a general-use method for a gene circuit 'voltmeter' or gene system algorithm 'print statement'. What is needed is a non-destructive, carrier non-modifying, means of testing 'live' biological systems at the single-molecule level. A method using the nanopore transduction detector (NTD) is demonstrated for single-molecule characterization in some situations, so may provide what is lacking. An important aspect of this approach is that use can be made of inexpensive antibody, protein, aptamer, duplex nucleic acid, or nucleic acid annealing molecules (for miRNA and viral monitoring) that have specific binding to the system component of interest. The NTD transducer's specific binding can also be designed to have low affinity binding as needed, such that there can be a 'catch and release' on low copy-number molecular components, such that there is not a disruption to the molecular system under study. NTD transducers are typically constructed by linking a binding moiety of interest to a 14 Stephen Winters-Hilt nanopore current modulator, where the modulator is designed to be electrophoretically drawn to the channel and partly captured, with its captured end distinctively modulating the flow of ions through the channel. Using inexpensive (commoditized) biomolecular components, such as DNA hairpins, this allows for an easily constructed, versatile, platform for biosensing. High specificity high affinity binding also allows a very versatile platform for assaying at the single molecule level, even down to the single isoform level, including molecular substructure profiling, such as glycosylation profiling in antibodies. An inexpensive commoditized pathway for constructing nanopore transducers is demonstrated. Nanopore transduction detector based reporter/event-transducer molecules may serve as a means to perform multicomponent mRNA-miRNA-protein and protein-protein systems analysis in general settings.

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Section: S3 Managing Common Interference Agents and Antibodies As Easupporting

confidence: 53%

Section: Managing Common Interference Agents and Aptamers And Antibomentioning

confidence: 99%

Section: Antibodies As Easily Identifiable Interference or Transducermentioning

confidence: 99%

Section: S1 Biotin-streptavidin Binding Studies Via Nanopore Event Tmentioning

confidence: 99%

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Biological system analysis using a nanopore transduction detector: from miRNA validation, to viral monitoring, to gene circuit feedback studies

Winters‐Hilt¹

2017

asms

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“…A single molecule’s blockading interaction upon capture in an ion channel can be self-modulating upon capture (i.e., without a dominant interaction state), and this has been found in a number of experiments [34-46]. The mechanism of interaction involves transient chemical bond formation between transducer and protein, where each bound state between transducer and channel imprints on the surrounding ionic current flow to provide a fixed level which then transitions to a different fixed level upon the bond dissociation or transitions to a different bound state.…”

Section: Introductionmentioning

confidence: 99%

The NTD Nanoscope: potential applications and implementations

2011

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BackgroundNanopore transduction detection (NTD) offers prospects for a number of highly sensitive and discriminative applications, including: (i) single nucleotide polymorphism (SNP) detection; (ii) targeted DNA re-sequencing; (iii) protein isoform assaying; and (iv) biosensing via antibody or aptamer coupled molecules. Nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. The NTD Nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. Meta Logos Inc. is developing both pre-packaged device platforms and component-level (unassembled) kit platforms (the latter described here). In both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. In this paper we provide an overview of the NTD Nanoscope applications and implementations. The NTD Nanoscope Kit, in particular, is a component-level reproduction of the standard NTD device used in previous research papers.ResultsThe NTD Nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. To expedite set-up in new lab settings, the calibration and troubleshooting for the NTD Nanoscope kit components and signal processing software, the NTD Nanoscope Kit, is designed to include a set of test buffers and control molecules based on experiments described in previous NTD papers (the model systems briefly described in what follows). The description of the Server-interfacing for advanced signal processing support is also briefly mentioned.ConclusionsSNP assaying, SNP discovery, DNA sequencing and RNA-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction (PCR) enzyme. The NTD Nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead.

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2021

Informatics and Machine Learning

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Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

Abstract: Background: The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.

Cited by 10 publications

References 33 publications

Biological system analysis using a nanopore transduction detector: from miRNA validation, to viral monitoring, to gene circuit feedback studies

Biological system analysis using a nanopore transduction detector: from miRNA validation, to viral monitoring, to gene circuit feedback studies

The NTD Nanoscope: potential applications and implementations

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