Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

JDRF nPOD-Virus Group

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Background: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10-1 to 10-8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10-8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10-7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10-6, while ISH detected the virus at dilutions of 10-4. Conclusions: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

Original languageEnglish (US)
Pages (from-to)21-28
Number of pages8
JournalJournal of Clinical Virology
Volume77
DOIs
StatePublished - Apr 1 2016

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Enterovirus
In Situ Hybridization
Mass Spectrometry
Immunohistochemistry
Polymerase Chain Reaction
Viruses
A549 Cells
Antibodies
Liquid Chromatography
Proteomics
Anti-Idiotypic Antibodies
Clone Cells
Peptides

Keywords

  • Immunohistochemisrty
  • In situ hybridization
  • Proteomics
  • RT-PCR
  • Sensitivity

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

@article{32478d2921e2413ab3f35405ac6c05d7,
title = "Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells",
abstract = "Background: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10-1 to 10-8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10-8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10-7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10-6, while ISH detected the virus at dilutions of 10-4. Conclusions: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.",
keywords = "Immunohistochemisrty, In situ hybridization, Proteomics, RT-PCR, Sensitivity",
author = "{JDRF nPOD-Virus Group} and Laiho, {Jutta E.} and Maarit Oikarinen and Richardson, {Sarah J.} and Gun Frisk and Julius Nyalwidhe and Burch, {Tanya C.} and Morris, {Margaret A.} and Sami Oikarinen and Alberto Pugliese and Francesco Dotta and Martha Campbell-Thompson and Jerry Nadler and Morgan, {Noel G.} and Heikki Hy{\"o}ty",
year = "2016",
month = "4",
day = "1",
doi = "10.1016/j.jcv.2016.01.015",
language = "English (US)",
volume = "77",
pages = "21--28",
journal = "Journal of Clinical Virology",
issn = "1386-6532",
publisher = "Elsevier",

}

TY - JOUR

T1 - Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

AU - JDRF nPOD-Virus Group

AU - Laiho, Jutta E.

AU - Oikarinen, Maarit

AU - Richardson, Sarah J.

AU - Frisk, Gun

AU - Nyalwidhe, Julius

AU - Burch, Tanya C.

AU - Morris, Margaret A.

AU - Oikarinen, Sami

AU - Pugliese, Alberto

AU - Dotta, Francesco

AU - Campbell-Thompson, Martha

AU - Nadler, Jerry

AU - Morgan, Noel G.

AU - Hyöty, Heikki

PY - 2016/4/1

Y1 - 2016/4/1

N2 - Background: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10-1 to 10-8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10-8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10-7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10-6, while ISH detected the virus at dilutions of 10-4. Conclusions: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

AB - Background: Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives: This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design: A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10-1 to 10-8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results: RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10-8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10-7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10-6, while ISH detected the virus at dilutions of 10-4. Conclusions: All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

KW - Immunohistochemisrty

KW - In situ hybridization

KW - Proteomics

KW - RT-PCR

KW - Sensitivity

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U2 - 10.1016/j.jcv.2016.01.015

DO - 10.1016/j.jcv.2016.01.015

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VL - 77

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JO - Journal of Clinical Virology

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