Titration von Human Coronaviren Mit einem Immunoperoxidase Assay

Published 4/28/2008
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Biology
 

ERRATUM NOTICE

Summary

In diesem Video zeigen wir eine alternative Methode für den Nachweis und Titrierung von Viren mit Hilfe einer enzymatischen Antigennachweis Technik als Immunperoxidase Test bekannt. Hier zeigen wir Ihnen, wie Sie Ihre virale Proben zu sammeln, bereiten die Zellen für die Prüfung und schließlich die Immunperoxidase Assay unter Verwendung von Verdünnungsreihen der virale Titer zu bestimmen.

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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

Berechnung von infektiösen viralen Titer stellt eine grundlegende und wichtige experimentelle Ansatz für Virologen. Klassische Plaque-Assays können nicht für Viren, die nicht verursachen erhebliche zytopathischen Effekte, die den Fall für Stämme 229E und OC43 der menschlichen Coronavirus (HCoV) verwendet werden. Eine alternative indirekte Immunperoxidase Assay (IPA) wird hier für den Nachweis und die Titration von diesen Viren beschrieben. Empfängliche Zellen mit seriellen Verdünnungen logarithmischen von Proben in einem 96-Well-Platte ausgesät. Nach viralen Wachstums ergibt Virusnachweis durch IPA das infektiöse Virus-Titer, als "Tissue Culture Infectious Dose" (TCID50) ausgedrückt. Dies entspricht der Verdünnung eines Virus-haltige Probe bei der die Hälfte einer Reihe von Labor-Brunnen enthalten replizierende Virus. Diese Technik ist eine zuverlässige Methode für die Titration von HCoV in biologischen Proben (Zellen, Gewebe oder Flüssigkeiten).

Protocol

Der vollständige Text Protokoll für diesen experimentellen Ansatz ist in Springer Protocols .

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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