scholarly journals Immunoprecipitation-targeted proteomics assays facilitate rational development of SARS-CoV-2 serological diagnostics

2021 ◽  
Author(s):  
Zhiqiang Fu ◽  
Yasmine Rais ◽  
Andrei Drabovich
Keyword(s):  
Infectious Diseases ◽  
Rational Design ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Selected Reaction Monitoring ◽  
Total Serum ◽  
Reaction Monitoring ◽  
Targeted Proteomics ◽  
Diagnostic Specificity ◽  
Serological Tests

Current design of serological tests employs conservative immunoassay approaches and is often focused on convenience, speed of manufacturing, and affordability. Limitations of such serological tests include semi-quantitative measurements, lack of standardization, potential cross-reactivity, and inability to distinguish between antibody subclasses. As a result of cross-reactivity, diagnostic specificity of serological antibody tests may not be sufficiently high to enable screening of the general asymptomatic populations for the acquired immunity against low-prevalence infectious diseases, such as COVID-19. Likewise, lack of a single standard for assay calibration limits inter-laboratory and international standardization of serological tests. In this study, we hypothesize that combination of immunoaffinity enrichments with targeted mass spectrometry measurements would enable rational design of serology diagnostics of infectious diseases, such as COVID-19. The same instrumental platform allows for sensitive and specific measurements of viral protein antigens, as wells as anti-viral antibodies circulating in human serum. Our proof-of-concept immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. In addition, a multiplex IP-selected reaction monitoring (IP-SRM) assay facilitated differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in patient sera. Simultaneous evaluation of numerous antigen-antibody subclass combinations revealed a receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Anti-RBD IgG1, IgG3, IgM and IgA1 subclasses, but not IgG2, IgG4 and IgA2, were found elevated in COVID-19-positive sera. Synthetic heavy isotope-labeled peptide internal standards as calibrators revealed elevated anti-RBD IgG1 in positive (510-6700 ng/mL; 0.02-0.22% of total serum IgG1) versus negative sera (60 [interquartile range 41-81] ng/mL). Likewise, anti-RBD IgM was elevated in positive (190-510 ng/mL; 0.06-0.16% of total serum IgM) versus negative sera (76 [31-108] ng/mL). Further validation of immunoprecipitation-targeted proteomics assays as a platform for serological assays will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer tools for comprehensive investigation of antibody isotype and subclass cooperation in immunity response.

Applying selected reaction monitoring to targeted proteomics

2011 ◽  
Vol 8 (2) ◽  
pp. 165-173 ◽  
Author(s):  
Enrique Calvo ◽  
Emilio Camafeita ◽  
Benjamín Fernández-Gutiérrez ◽  
Juan A López
Keyword(s):  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Proteomics

scholarly journals Validation of an Enzyme Immunoassay for Detection and Semiquantification of Cannabinoids in Oral Fluid

2010 ◽  
Vol 56 (6) ◽  
pp. 1007-1014 ◽  
Author(s):  
David M Schwope ◽  
Garry Milman ◽  
Marilyn A Huestis
Keyword(s):  
Oral Fluid ◽  
Drugs Of Abuse ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Driving Under The Influence ◽  
Diagnostic Efficiency ◽  
Diagnostic Specificity ◽  
Collection Device ◽  
Assay Performance ◽  
Direct Elisa

Abstract Background: Oral fluid (OF) is gaining prominence as an alternative matrix for monitoring drugs of abuse in the workplace, criminal justice, and driving under the influence of drugs programs. It is important to characterize assay performance and limitations of screening techniques for Δ9-tetrahydrocannabinol (THC) in OF. Methods: We collected OF specimens by use of the Quantisal™ OF collection device from 13 daily cannabis users after controlled oral cannabinoid administration. All specimens were tested with the Immunalysis Sweat/OF THC Direct ELISA and confirmed by 2-dimensional GC-MS. Results: The limit of detection was <1 μg/L THC equivalent, and the assay demonstrated linearity from 1 to 50 μg/L, with semiquantification to 200 μg/L. Intraplate imprecision (n = 7) ranged from 2.9% to 7.7% CV, and interplate imprecision (n = 20) was 3.0%–9.1%. Cross-reactivities at 4 μg/L were as follows: 11-hydroxy-THC, 198%; Δ8-tetrahydrocannabinol (Δ8-THC), 128%; 11-nor-9-carboxy-THC (THCCOOH), 121%; THC (target), 98%; cannabinol, 87%; THCCOOH-glucuronide, 11%; THC-glucuronide, 10%; and cannabidiol, 2.4%. Of 499 tested OF specimens, 52 confirmed positive (THC 2.0–290 μg/L), with 100% diagnostic sensitivity at the proposed Substance Abuse and Mental Health Services Administration screening cutoff of 4 μg/L cannabinoids and GC-MS cutoff of 2 μg/L THC. Forty-seven specimens screened positive but were not confirmed by 2D-GC-MS, yielding 89.5% diagnostic specificity and 90.6% diagnostic efficiency. Thirty-one of 47 unconfirmed immunoassay positive specimens were from 1 individual and contained >400 ng/L THCCOOH, potentially contributing to cross-reactivity. Conclusions: The Immunalysis Sweat/OF THC Direct ELISA is an effective screening procedure for detecting cannabinoids in OF.

scholarly journals The development of selected reaction monitoring methods for targeted proteomics via empirical refinement

PROTEOMICS ◽  
2012 ◽  
Vol 12 (8) ◽  
pp. 1134-1141 ◽  
Author(s):  
Michael S. Bereman ◽  
Brendan MacLean ◽  
Daniela M. Tomazela ◽  
Daniel C. Liebler ◽  
Michael J. MacCoss
Keyword(s):  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Proteomics ◽  
Monitoring Methods

Screening Method Using Selected Reaction Monitoring for Targeted Proteomics Studies of Nasal Lavage Fluid

2012 ◽  
Vol 12 (1) ◽  
pp. 234-247 ◽  
Author(s):  
Harriet Mörtstedt ◽  
Monica H. Kåredal ◽  
Bo A. G. Jönsson ◽  
Christian H. Lindh
Keyword(s):  
Screening Method ◽  
Lavage Fluid ◽  
Nasal Lavage ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Proteomics ◽  
Nasal Lavage Fluid

High-throughput production of a stable isotope-labeled peptide library for targeted proteomics using a wheat germ cell-free synthesis system

2016 ◽  
Vol 12 (8) ◽  
pp. 2389-2393 ◽  
Author(s):  
Nobuaki Takemori ◽  
Ayako Takemori ◽  
Yuki Tanaka ◽  
Jun Ishizaki ◽  
Hitoshi Hasegawa ◽  
...  
Keyword(s):  
High Throughput ◽  
Wheat Germ ◽  
Peptide Library ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Proteomics ◽  
Synthesis System ◽  
System P ◽  
Reference Peptide ◽  
Protein Synthesis System

Development of a reference peptide library for selected reaction monitoring (SRM)-based targeted proteomics using a high-throughput protein synthesis system.

scholarly journals Parallel Reaction Monitoring for High Resolution and High Mass Accuracy Quantitative, Targeted Proteomics

2012 ◽  
Vol 11 (11) ◽  
pp. 1475-1488 ◽  
Author(s):  
Amelia C. Peterson ◽  
Jason D. Russell ◽  
Derek J. Bailey ◽  
Michael S. Westphall ◽  
Joshua J. Coon
Keyword(s):  
High Resolution ◽  
Dynamic Range ◽  
High Mass ◽  
Accurate Mass ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Proteomics ◽  
Triple Quadrupole ◽  
Parallel Reaction ◽  
Parallel Reaction Monitoring

Selected reaction monitoring on a triple quadrupole mass spectrometer is currently experiencing a renaissance within the proteomics community for its, as yet, unparalleled ability to characterize and quantify a set of proteins reproducibly, completely, and with high sensitivity. Given the immense benefit that high resolution and accurate mass instruments have brought to the discovery proteomics field, we wondered if highly accurate mass measurement capabilities could be leveraged to provide benefits in the targeted proteomics domain as well. Here, we propose a new targeted proteomics paradigm centered on the use of next generation, quadrupole-equipped high resolution and accurate mass instruments: parallel reaction monitoring (PRM). In PRM, the third quadrupole of a triple quadrupole is substituted with a high resolution and accurate mass mass analyzer to permit the parallel detection of all target product ions in one, concerted high resolution mass analysis. We detail the analytical performance of the PRM method, using a quadrupole-equipped bench-top Orbitrap MS, and draw a performance comparison to selected reaction monitoring in terms of run-to-run reproducibility, dynamic range, and measurement accuracy. In addition to requiring minimal upfront method development and facilitating automated data analysis, PRM yielded quantitative data over a wider dynamic range than selected reaction monitoring in the presence of a yeast background matrix because of PRM's high selectivity in the mass-to-charge domain. With achievable linearity over the quantifiable dynamic range found to be statistically equal between the two methods, our investigation suggests that PRM will be a promising new addition to the quantitative proteomics toolbox.

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