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Find video protocols related to scientific articles indexed in Pubmed.
The submerged printing of cells onto a modified surface using a continuous flow microspotter.
J Vis Exp
PUBLISHED: 05-07-2014
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The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.
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High-throughput epitope binning of therapeutic monoclonal antibodies: why you need to bin the fridge.
Drug Discov. Today
PUBLISHED: 02-27-2014
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Analytical tools are evolving to meet the need for the higher-throughput characterization of therapeutic monoclonal antibodies. An antibody's epitope is arguably its most important property because it underpins its functional activity but, because epitope selection is innate, it remains an empirical process. Here, we focus on the emergence of label-free biosensors with throughput capabilities orders of magnitude higher than the previous state-of-the-art, which can facilitate large assays such as epitope binning so that they can be incorporated alongside functional activity screens, enabling the rapid identification of leads that exhibit unique and functional epitopes. In addition to streamlining the drug development process by saving time and cost, the information from epitope binning assays could provide the basis for intellectual property protection.
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A critical comparison of protein microarray fabrication technologies.
Analyst
PUBLISHED: 01-31-2014
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Of the diverse analytical tools used in proteomics, protein microarrays possess the greatest potential for providing fundamental information on protein, ligand, analyte, receptor, and antibody affinity-based interactions, binding partners and high-throughput analysis. Microarrays have been used to develop tools for drug screening, disease diagnosis, biochemical pathway mapping, protein-protein interaction analysis, vaccine development, enzyme-substrate profiling, and immuno-profiling. While the promise of the technology is intriguing, it is yet to be realized. Many challenges remain to be addressed to allow these methods to meet technical and research expectations, provide reliable assay answers, and to reliably diversify their capabilities. Critical issues include: (1) inconsistent printed microspot morphologies and uniformities, (2) low signal-to-noise ratios due to factors such as complex surface capture protocols, contamination, and static or no-flow mass transport conditions, (3) inconsistent quantification of captured signal due to spot uniformity issues, (4) non-optimal protocol conditions such as pH, temperature, drying that promote variability in assay kinetics, and lastly (5) poor protein (e.g., antibody) printing, storage, or shelf-life compatibility with common microarray assay fabrication methods, directly related to microarray protocols. Conventional printing approaches, including contact (e.g., quill and solid pin), non-contact (e.g., piezo and inkjet), microfluidics-based, microstamping, lithography, and cell-free protein expression microarrays, have all been used with varying degrees of success with figures of merit often defined arbitrarily without comparisons to standards, or analytical or fiduciary controls. Many microarray performance reports use bench top analyte preparations lacking real-world relevance, akin to "fishing in a barrel", for proof of concept and determinations of figures of merit. This review critiques current protein-based microarray preparation techniques commonly used for analytical and function-based proteomics and their effects on array-based assay performance.
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High-throughput epitope binning assays on label-free array-based biosensors can yield exquisite epitope discrimination that facilitates the selection of monoclonal antibodies with functional activity.
PLoS ONE
PUBLISHED: 01-01-2014
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Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.
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Continuous scaling 3d micro flow printing for improved spot morphology in protein microarrays - biomed 2013.
Biomed Sci Instrum
PUBLISHED: 05-21-2013
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The protein microarray platform while innovative still poses a number of challenges which can only be met through creative and sophisticated system design. Pin printing while allowing for flexibility as to the type of medium printed does not offer the kind of spot reproducibility that a very sensitive application may require. The Continuous Flow Microspotter (CFM) was designed to not only allow for flexibility and reproducibility but to also achieve solution stability through flow scaling. This study uses the emerging CFM for printing protein and antibodies three dimensionally for general protein microarray applications. Consistent spot morphology, a continual and persistent problem in traditional pin printed microarrays, was compared under variable printed flow rates. The final assessment was performed using a rudimentary shear model. Force effects discussion and statistical data was used to demonstrate the versatility of the system.
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Sensitivity of protein array deposition using continuous flow printing for fluorescent microarray applications - biomed 2013.
Biomed Sci Instrum
PUBLISHED: 05-21-2013
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The promise of antibody and protein microarrays to revolutionize disease diagnostics has failed to live up to the hype primarily due to the problems associated with the printing of the antibodies and/or proteins onto the detection surface. The current standard in printing proteins is pin printing. An alternative to the pin printer is the continuous-flow microspotter (CFM), a protein printer that uses microfluidic flow to print down the proteins. The advantages of the CFM include consistent spot morphology, spot-to-spot uniformity and enhanced surface concentration. Further, the CFM is effective at capturing proteins and antibodies from either dilute or complex (e.g. blood or tissue) samples. In this study, the sensitivity of CFM printing Cy3 and Cy5 fluorescently labeled proteins was determined. Values were obtained at low concentrations tens of ng/mL with low coefficients of variation. Thus, the CFM can effectively print and quantify proteins and antibodies from low concentration and complex buffered samples.
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Improved biomolecule microarrays by printing on nanoporous aluminum oxide using a continuous-flow microspotter.
Small
PUBLISHED: 06-22-2010
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Biomolecules, including protein A, albumin, and immunoglobulin G, are spotted on top of a nanoporous substrate by using a continuous-flow microspotter (CFM) system, which normally produces spots 3 to 4 orders of magnitude more sensitive than conventional biomolecule printing methods. The spots are observed with a fluorescence scanner. By using the CFM to print spots on nanoporous substrates, an additional order of magnitude increase in signal is observed, which leads to high signal-to-background ratios, highly saturated spots, and a measurable signal at printing concentrations as low as 1.6 ng mL(-1). This technique produces highly concentrated biomolecular spots from dilute samples and significantly increases the sensitivity of sensing platforms.
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In situ microarray fabrication and analysis using a microfluidic flow cell array integrated with surface plasmon resonance microscopy.
Anal. Chem.
PUBLISHED: 05-05-2009
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Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.
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Detergent screening of a G-protein-coupled receptor using serial and array biosensor technologies.
Anal. Biochem.
PUBLISHED: 01-13-2009
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We describe the benefits and limitations of two biosensor approaches for screening solubilization conditions for G-protein-coupled receptors (GPCRs). Assays designed for a serial processing instrument (Biacore 2000/3000/T100) and an array platform (Biacore Flexchip) were used to examine how effectively 96 different detergents solubilized the chemokine receptor CCR5 while maintaining its binding activity for a conformationally sensitive Fab (2D7). Using the serial processing instrument, we were able to analyze three samples in each 30-min binding cycle, thereby requiring approximately 24h to screen an entire 96-well plate of conditions. In-line capturing allowed us to normalize the 2D7 binding responses for different receptor capture levels. In contrast, with the array system, we could characterize the effects of all 96 detergents simultaneously, completing the assay in less than 1h. But the current array technology requires that we capture the GPCR preparations off-line, making it more challenging to normalize for receptor capture levels. Also, the array platform is less sensitive than the serial platforms, thereby limiting the size of the analyte to larger molecules (>5000Da). Overall, the two approaches proved to be highly complementary; both assays identified identical detergents that produced active solubilized CCR5 as well as those detergents that either were ineffective solubilizers or inactivated the receptor.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.