scholarly journals Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

2021 ◽  
Author(s):  
Angela Mc Ardle ◽  
Aleksandra Binek, ◽  
Annie Moradian ◽  
Blandine Chazarin Orgel ◽  
Alejandro Rivas ◽  
...  
Keyword(s):  
High Throughput ◽  
Whole Blood ◽  
Large Scale ◽  
Dynamic Range ◽  
Profile Analysis ◽  
Protein Biomarkers ◽  
Throughput Analysis ◽  
Broad Dynamic Range ◽  
Plasma Fractions ◽  
Deep Profile

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid–throughput analysis for three blood–based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA–MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high–throughput or deep profile analysis with mid–throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5—6–point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high–throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid–throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

scholarly journals Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics

2015 ◽  
Vol 112 (32) ◽  
pp. E4354-E4363 ◽  
Author(s):  
Fatih Inci ◽  
Chiara Filippini ◽  
Murat Baday ◽  
Mehmet Ozgun Ozen ◽  
Semih Calamak ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Point Of Care ◽  
Medical Diagnostics ◽  
Clinical Samples ◽  
Label Free ◽  
Protein Biomarkers ◽  
Electrical Field ◽  
Color Changes ◽  
Sensing Platform ◽  
Broad Dynamic Range

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients’ homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE2RD), which addresses all these impediments on a single platform. The NE2RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE2RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE2RD’s broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients’ homes.

scholarly journals Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Rutger M. Schepp ◽  
Cornelis A. M. de Haan ◽  
Deidre Wilkins ◽  
Hans Layman ◽  
Barney S. Graham ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
High Throughput ◽  
Large Scale ◽  
Dynamic Range ◽  
Valuable Insight ◽  
Conformational Structure ◽  
Multiplex Immunoassay ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Antibody Levels

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies. IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

Genetics ◽  
2021 ◽  
Author(s):  
Reine U Protacio ◽  
Tresor O Mukiza ◽  
Mari K Davidson ◽  
Wayne P Wahls
Keyword(s):  
Fission Yeast ◽  
Frequency Distribution ◽  
Meiotic Recombination ◽  
Environmental Conditions ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Dynamic Range ◽  
Recombination Rates ◽  
Isolated Populations ◽  
Broad Dynamic Range

Abstract It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.

scholarly journals Shotgun Proteomics and Biomarker Discovery

2002 ◽  
Vol 18 (2) ◽  
pp. 99-105 ◽  
Author(s):  
W. Hayes McDonald ◽  
John R. Yates
Keyword(s):  
Large Scale ◽  
Protein Identification ◽  
Biomarker Discovery ◽  
Dynamic Range ◽  
Shotgun Proteomics ◽  
Sequence Information ◽  
Protein Biomarkers ◽  
Post Translational Modifications ◽  
Multidimensional Protein Identification Technology ◽  
Shotgun Approach

Coupling large-scale sequencing projects with the amino acid sequence information that can be gleaned from tandem mass spectrometry (MS/MS) has made it much easier to analyze complex mixtures of proteins. The limits of this “shotgun” approach, in which the protein mixture is proteolytically digested before separation, can be further expanded by separating the resulting mixture of peptides prior to MS/MS analysis. Both single dimensional high pressure liquid chromatography (LC) and multidimensional LC (LC/LC) can be directly interfaced with the mass spectrometer to allow for automated collection of tremendous quantities of data. While there is no single technique that addresses all proteomic challenges, the shotgun approaches, especially LC/LC-MS/MS-based techniques such as MudPIT (multidimensional protein identification technology), show advantages over gel-based techniques in speed, sensitivity, scope of analysis, and dynamic range. Advances in the ability to quantitate differences between samples and to detect for an array of post-translational modifications allow for the discovery of classes of protein biomarkers that were previously unassailable.

scholarly journals Remote fingerstick blood collection for SARS-CoV-2 antibody testing

2020 ◽  
Author(s):  
Wilfredo F. Garcia-Beltran ◽  
Tyler E. Miller ◽  
Grace Kirkpatrick ◽  
Andrea Nixon ◽  
Michael G. Astudillo ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Dynamic Range ◽  
Rapid Development ◽  
Venous Blood ◽  
Blood Collection ◽  
Antibody Assay ◽  
Serum Samples ◽  
Blood Draw ◽  
Serological Tests

ABSTACT The rapid worldwide spread of severe acute respiratory system coronavirus 2 (SARSCoV-2) infection has propelled the rapid development of serological tests that can detect anti-SARS-CoV-2 antibodies. These have been used for studying the prevalence and spread of infection in different populations, helping establish a recent diagnosis of coronavirus disease 2019 (COVID-19), and will likely be used to confirm humoral immunity after infection or vaccination. However, nearly all lab-based high-throughput SARS-CoV-2 serological assays require a serum sample from venous blood draw, limiting their applications and scalability. Here, we present a method that enables large scale SARS-CoV-2 serological studies by combining self or office collection of fingerprick blood with a volumetric absorptive microsampling device (Mitra, Neoteryx, LLC) with a high-throughput electrochemiluminescence-based SARS-CoV-2 total antibody assay (Roche Elecsys, Roche Diagnostics, Inc.) that is emergency use authorization (EUA) approved for use on serum samples and widely used by clinical laboratories around the world. We found that the Roche Elecsys assay has a high dynamic range that allows for accurate detection of SARS-CoV-2 antibodies in serum samples diluted 1:20 as well as contrived dried blood extracts. Extracts of dried blood from Mitra devices acquired in a community seroprevalence study showed near identical sensitivity and specificity in detection of SARS-CoV-2 antibodies as compared to neat sera using predefined thresholds for each specimen type. Overall, this study affirms the use of Mitra dried blood collection device with the Roche Elecsys SARS-CoV-2 total antibody assay for remote or at-home testing as well as large-scale community seroprevalence studies.

scholarly journals FRET-Seq: a High-Throughput FRET-Based Screening Platform to Improve FRET Biosensors in Mammalian Cells

2021 ◽  
Author(s):  
Longwei Liu ◽  
Praopim Limsakul ◽  
Xianhui Meng ◽  
Yan Huang ◽  
Reed E. S. Harrison ◽  
...  
Keyword(s):  
High Throughput ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Large Scale ◽  
Dynamic Range ◽  
Spatiotemporal Resolution ◽  
Fret Biosensors ◽  
Screening Platform ◽  
Sensitivity Specificity ◽  
Generation Sequencing

Abstract Genetically-encoded biosensors based on FRET have been widely used to dynamically monitor the activity of protein tyrosine kinases (PTKs) in living cell with high spatiotemporal resolution. However, the limitation in sensitivity, specificity, and dynamic range of FRET biosensors have hindered their broader applications. Here, we introduced a systematic platform, FRET-Seq, which integrates high-throughput FRET sorting and next-generation sequencing, to identify FRET biosensors with better performance from large-scale libraries directly in mammalian cells.

scholarly journals High-throughput Analysis of Arabidopsis Stem Vibrations to Identify Mutants with Altered Mechanical Properties

2018 ◽  
Author(s):  
Miyuki T. Nakata ◽  
Masahiro Takahara ◽  
Shingo Sakamoto ◽  
Kouki Yoshida ◽  
Nobutaka Mitsuda
Keyword(s):  
Mechanical Properties ◽  
High Throughput ◽  
High Speed ◽  
Large Scale ◽  
Growth Traits ◽  
Damping Ratio ◽  
Free Vibrations ◽  
Video Data ◽  
High Throughput Analysis ◽  
Throughput Analysis

AbstractMechanical properties are rarely used as quantitative indices for the large-scale mutant screening of plants, even in the model plant Arabidopsis thaliana. The mechanical properties of plant stems generally influence their vibrational characteristics. Here, we developed Python-based software, named AraVib, for the high-throughput analysis of free vibrations of plant stems, focusing specifically on Arabidopsis stem vibrations, and its extended version, named AraVibS, to identify mutants with altered mechanical properties. These programs can be used without knowledge of Python and require only an inexpensive handmade setting stand and an iPhone/iPad with a high-speed shooting function for data acquisition. Using our system, we identified an nst1 nst3 double-mutant lacking secondary cell walls in fiber cells and a wrky12 mutant displaying ectopic formation of secondary cell wall compared with wild type by employing only two growth traits (stem height and fresh weight) in addition to videos of stem vibrations. Furthermore, we calculated the logarithmic decrement, the damping ratio, the natural frequency and the stiffness based on the spring-mass-damper model from the video data using AraVib. The stiffness was estimated to be drastically decreased in nst1 nst3, which agreed with previous tensile test results. However, in wrky12, the stiffness was significantly increased. These results demonstrate the effectiveness of our new system. Because our method can be applied in a high-throughput manner, it can be used to screen for mutants with altered mechanical properties.

scholarly journals Multiplexed micronutrient, inflammation, and malarial antigenemia assessment using a plasma fractionation device

2021 ◽  
Author(s):  
Eleanor Brindle ◽  
Lorriane L. Lillis ◽  
Rebecca Barney ◽  
Pooja Bansil ◽  
Francisco Arredondo ◽  
...  
Keyword(s):  
Serum Ferritin ◽  
Whole Blood ◽  
Large Scale ◽  
Disease Status ◽  
Dried Blood Spots ◽  
Micronutrient Deficiencies ◽  
Protein Biomarkers ◽  
Micronutrient Status ◽  
Highly Correlated ◽  
Finger Stick

Collecting, processing, and storing blood samples for future analysis of biomarkers can be challenging when performed in resource limited environments. The preparation of dried blood spots (DBS) from heel or finger stick collection of whole blood is a widely used and established method. DBS pose less risk of infection from blood borne pathogens, do not require immediate specimen processing and tolerate a wider range of storage temperatures, and are easier to ship. As such, DBS are commonly used in large-scale surveys to assess infectious disease status and/or micronutrient status in vulnerable populations. Recently, we reported that DBS can be used with a multiplexed immunoassay, the Q-plex Human Micronutrient 7-plex Array (MN 7-plex). This tool can simultaneously quantify seven protein biomarkers related to micronutrient deficiencies (iodine, iron and vitamin A), inflammation and malarial antigenemia using plasma or serum. Serum ferritin, a key iron biomarker, cannot be measured from DBS due to red blood cell (RBC) ferritin confounding the results. In this study, we demonstrate the performance of a simple and rapid blood fractionation tool that passively separates serum from cellular components via diffusion through a membrane into a plasma collection disc (PCD) to produce plasma spots. We evaluated the concordance of MN 7-plex analyte concentrations from matched panels of eighty-eight samples of PCD, DBS, and wet plasma prepared from anticoagulated venous whole blood. The results show high correlation between eluates from PCD and DBS and wet plasma for each analyte. Serum ferritin measures from the PCD eluates were highly correlated to wet plasma samples. This suggests that surveillance for iron deficiency may be improved over the current methods restricted to only measuring sTfR in DBS as when used in combination with the MN 7-plex, all seven biomarkers can be simultaneously measured using PCDs.

scholarly journals A high-throughput microfluidic nano-immunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultra-low volume dried blood samples

2020 ◽  
Author(s):  
Zoe Swank ◽  
Grégoire Michielin ◽  
Hon Ming Yip ◽  
Patrick Cohen ◽  
Diego O. Andrey ◽  
...  
Keyword(s):  
High Throughput ◽  
Whole Blood ◽  
Large Scale ◽  
Test Strip ◽  
Sample Collection ◽  
Serum Samples ◽  
Blood Samples ◽  
Novel Technologies ◽  
Glucose Test ◽  
Low Volume

AbstractNovel technologies are needed to facilitate large-scale detection and quantification of SARS-CoV-2 specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies, vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nano-immunnoassay for the detection of anti-SARS-CoV-2 IgG antibodies in 1024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultra-low volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL whole blood easily obtainable from a simple fingerprick. The nano-immunoassay platform achieves high-throughput, high sensitivity and specificity, negligible reagent consumption, and a decentralized and simple approach to blood sample collection. We expect this technology to be immediately applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker diagnostics in general.

Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood

2010 ◽  
Vol 104 (08) ◽  
pp. 392-401 ◽  
Author(s):  
Katleen Broos ◽  
Alexandre Fontayne ◽  
Tímea Szántó ◽  
Changgeng Ruan ◽  
Alan Nurden ◽  
...  
Keyword(s):  
Platelet Function ◽  
High Throughput ◽  
Whole Blood ◽  
Large Scale ◽  
Platelet Rich Plasma ◽  
Sandwich Elisa ◽  
Elisa Assay ◽  
Platelet Response ◽  
Healthy Donors ◽  
Diagnostic Values

SummaryPlatelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or αIIbβ3 and GPIbα were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

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