Titrering av mänskliga coronavirus Använda en immunofluorescensteknik analys

Published 4/28/2008
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Biology
 

ERRATUM NOTICE

Summary

I denna video visar vi en alternativ metod för att upptäcka och titering av virus med hjälp av en enzymatisk metod för påvisande av antigen som kallas en immunofluorescensteknik analys. Här kommer vi att visa dig hur du hämta din virala prover, förbereda celler för att testa, och slutligen immunofluorescensteknik analysen med seriella spädningar för att bestämma virus titer.

Cite this Article

Copy Citation

Lambert, F., Jacomy, H., Marceau, G., J. Talbot, P. Titration of Human Coronaviruses Using an Immunoperoxidase Assay. J. Vis. Exp. (14), e751, doi:10.3791/751 (2008).

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Abstract

Beräkning av smittsam virussjukdom titrar är en grundläggande och viktiga experimentell metod för virologer. Klassisk plack analyser kan inte användas för virus som inte orsakar betydande cytopatisk effekter, vilket är fallet för stammar 229E och OC43 av mänskliga coronavirus (HCoV). Ett alternativ indirekt immunofluorescensteknik analys (IPA), vilka beskrivs nedan för upptäckt och titrering av dessa virus. Mottagliga celler inokuleras med seriell logaritmisk spädningar av prover i ett 96-brunnar. Efter viral tillväxt, ger virus upptäckt av IPA den smittsamma virus titer, uttryckt som "vävnadsodling infektionsdos" (TCID50). Detta representerar utspädning av ett virus som innehåller prov vid vilken hälften av en serie av laboratorie-brunnar innehåller replikera virus. Denna teknik är en tillförlitlig metod för titrering av HCoV i biologiska prover (celler, vävnader eller vätskor).

Protocol

Den fullständiga texten protokollet för denna experimentella strategi finns i Springer protokoll .

Erratum

Formal Correction: Erratum: Titration of Human Coronaviruses Using an Immunoperoxidase Assay
Posted by JoVE Editors on 04/01/2012. Citeable Link.

A correction was made to: Titration of Human Coronaviruses Using an Immunoperoxidase Assay. A revised abstract was republished due to a publisher error.

Revised Abstract:

Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.

Original Abstract:

Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues or fluids).

Comments

3 Comments

  1. Nice job!!!

    We would like to titer OC43 coronavirus strain and we don't have specific antibodies, do you have any alternative solution for titering it.

    Thank you for your consideration

    Gael Belliot, PhD

    Laboratory of Virology
    CHU Dijon France

    Reply
    Posted by: Anonymous
    July 3, 2009 - 11:11 AM
  2. Unfortunately, as the classical plaque assays cannot be used for human coronavirus OC-43, you have to use antibodies for IP detection (some are available commercially).

    Sincerely

    Helene jacomy

    Reply
    Posted by: Anonymous
    July 3, 2009 - 1:53 PM
  3. Thanks

    GB

    Reply
    Posted by: Anonymous
    July 12, 2009 - 3:23 PM

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