Towards a human proteome atlas: High-throughput generation of mono-specific antibodies for tissue profiling

PROTEOMICS ◽  
2005 ◽  
Vol 5 (17) ◽  
pp. 4327-4337 ◽  
Author(s):  
Peter Nilsson ◽  
Linda Paavilainen ◽  
Karin Larsson ◽  
Jenny Ödling ◽  
Mårten Sundberg ◽  
...  
Keyword(s):  
High Throughput ◽  
Human Proteome ◽  
Specific Antibodies ◽  
Tissue Profiling

scholarly journals An Optimized High-Throughput Neutralization Assay for Hepatitis E Virus (HEV) Involving Detection of Secreted Porf2

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 64 ◽  
Author(s):  
Chang Liu ◽  
Wei Cai ◽  
Xin Yin ◽  
Zimin Tang ◽  
Guiping Wen ◽  
...  
Keyword(s):  
High Throughput ◽  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Neutralization Assay ◽  
The Novel ◽  
Specific Antibodies ◽  
Neutralizing Activity ◽  
Elisa Kit ◽  
Neutralizing Capacity ◽  
Potential Threshold

Hepatitis E virus (HEV) is a common cause of acute hepatitis worldwide. Current methods for evaluating the neutralizing activity of HEV-specific antibodies include immunofluorescence focus assays (IFAs) and real-time PCR, which are insensitive and operationally complicated. Here, we developed a high-throughput neutralization assay by measuring secreted pORF2 levels using an HEV antigen enzyme-linked immunosorbent assay (ELISA) kit based on the highly replicating HEV genotype (gt) 3 strain Kernow. We evaluated the neutralizing activity of HEV-specific antibodies and the sera of vaccinated individuals (n = 15) by traditional IFA and the novel assay simultaneously. A linear regression analysis shows that there is a high degree of correlation between the two assays. Furthermore, the anti-HEV IgG levels exhibited moderate correlation with the neutralizing titers of the sera of vaccinated individuals, indicating that immunization with gt 1 can protect against gt 3 Kernow infection. We then determined specificity of the novel assay and the potential threshold of neutralizing capacity using anti-HEV IgG positive sera (n = 27) and anti-HEV IgG negative sera (n = 23). The neutralizing capacity of anti-HEV IgG positive sera was significantly stronger than that of anti-HEV IgG negative. In addition, ROC curve analysis shows that the potential threshold of neutralizing capacity of sera was 8.07, and the sensitivity and specificity of the novel assay was 88.6% and 100%, respectively. Our results suggest that the neutralization assay using the antigen ELISA kit could be a useful tool for HEV clinical research.

From genes to proteins: High-throughput expression and purification of the human proteome

2000 ◽  
Vol 80 (2) ◽  
pp. 187-191 ◽  
Author(s):  
Joanna S. Albala ◽  
Ken Franke ◽  
Ian R. McConnell ◽  
Karen L. Pak ◽  
Peg A. Folta ◽  
...  
Keyword(s):  
High Throughput ◽  
Human Proteome ◽  
Expression And Purification

scholarly journals An Optimized High-Throughput Immuno-Plaque Assay for SARS-CoV-2

2021 ◽  
Vol 12 ◽  
Author(s):  
Alberto A. Amarilla ◽  
Naphak Modhiran ◽  
Yin Xiang Setoh ◽  
Nias Y. G. Peng ◽  
Julian D. J. Sng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Throughput ◽  
Plaque Assay ◽  
Neutralization Assay ◽  
Great Flexibility ◽  
Specific Antibodies ◽  
Research Outcomes ◽  
Global Spread ◽  
Rapid Emergence ◽  
Detection And Quantification

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

scholarly journals High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

2020 ◽  
Author(s):  
Sai Priya Anand ◽  
Jérémie Prévost ◽  
Jonathan Richard ◽  
Josée Perreault ◽  
Tony Tremblay ◽  
...  
Keyword(s):  
High Throughput ◽  
Viral Entry ◽  
293T Cell ◽  
Full Length ◽  
Strongly Correlated ◽  
Specific Antibodies ◽  
Main Target ◽  
High Throughput Detection ◽  
Humoral Responses ◽  
Design And Methods

AbstractBackgroundThe SARS-CoV-2 virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than a million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection.Study Design and MethodsTo extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike specific antibodies in the plasma of convalescent donors.Results and ConclusionWe found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations, but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

Proteomic Cinderella: Customized analysis of bulky MS/MS data in one night

2018 ◽  
Vol 16 (01) ◽  
pp. 1740011 ◽  
Author(s):  
Olga Kiseleva ◽  
Ekaterina Poverennaya ◽  
Alexander Shargunov ◽  
Andrey Lisitsa
Keyword(s):  
User Interface ◽  
Open Source ◽  
High Throughput ◽  
Graphical User Interface ◽  
Modern Science ◽  
Human Proteome ◽  
Tandem Ms ◽  
Report Generation ◽  
Manual Search ◽  
Human Proteins

Proteomic challenges, stirred up by the advent of high-throughput technologies, produce large amount of MS data. Nowadays, the routine manual search does not satisfy the “speed” of modern science any longer. In our work, the necessity of single-thread analysis of bulky data emerged during interpretation of HepG2 proteome profiling results for proteoforms searching. We compared the contribution of each of the eight search engines (X!Tandem, MS-GF[Formula: see text], MS Amanda, MyriMatch, Comet, Tide, Andromeda, and OMSSA) integrated in an open-source graphical user interface SearchGUI ( http://searchgui.googlecode.com ) into total result of proteoforms identification and optimized set of engines working simultaneously. We also compared the results of our search combination with Mascot results using protein kit UPS2, containing 48 human proteins. We selected combination of X!Tandem, MS-GF[Formula: see text] and OMMSA as the most time-efficient and productive combination of search. We added homemade java-script to automatize pipeline from file picking to report generation. These settings resulted in rise of the efficiency of our customized pipeline unobtainable by manual scouting: the analysis of 192 files searched against human proteome (42153 entries) downloaded from UniProt took 11[Formula: see text]h.

Cancer biology through immunohistology

2019 ◽  
pp. 394-409
Author(s):  
Karen Pulford ◽  
Kevin Gatter
Keyword(s):  
Image Analysis ◽  
Microscopic Study ◽  
High Throughput ◽  
Cancer Biology ◽  
Critical Component ◽  
Predictive Information ◽  
Specific Antibodies ◽  
Essential Tool ◽  
Cellular Components

Immunohistology is the microscopic study of cells and tissues using specific antibodies that bind to individual molecules expressed by the cellular and non-cellular components of the tissues. This branch of science is an essential link in the analysis and interpretation of data from high throughput genomic and proteomic technologies. Its use, both in the research and in the clinical arenas, has led to an increased understanding of cancer biology. This knowledge has also resulted in improvements in diagnosis, the provision of prognostic and predictive information, and highlighted the use of appropriate treatments. Furthermore, immunohistochemistry is a critical component in the search for personalized treatments. The ongoing advances in the availability of specific validated antibodies, continued improvements in staining and image analysis, and the integration of different technologies will ensure that immunohistochemistry becomes an even more essential tool in the study of cancer biology.

scholarly journals High-Throughput Identification of Mammalian Secreted Proteins Using Species-Specific Scheme and Application to Human Proteome

Molecules ◽  
2018 ◽  
Vol 23 (6) ◽  
pp. 1448 ◽  
Author(s):  
Jian Zhang ◽  
Haiting Chai ◽  
Song Guo ◽  
Huaping Guo ◽  
Yanling Li
Keyword(s):  
High Throughput ◽  
Human Proteome ◽  
Secreted Proteins ◽  
Specific Scheme ◽  
Species Specific
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