Le titrage de coronavirus humains en utilisant un dosage immunoperoxydase

Published 4/28/2008
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Biology
 

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Summary

Dans cette vidéo, nous démontrons une méthode alternative de détection et de titrage de virus en utilisant une technique de détection d'antigènes enzymatique connu sous le nom d'un dosage immuno. Ici, nous allons vous montrer comment collecter vos échantillons viraux, préparer les cellules pour les tests, et enfin le dosage immuno utilisant des dilutions en série pour déterminer le titre viral.

Cite this Article

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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

Calcul des titres viraux infectieux représente une approche fondamentale et essentielle expérimental pour virologues. Dosages de plaque classique ne peut pas être utilisé pour des virus qui ne provoquent pas des effets cytopathiques, qui est le cas pour les souches 229E et OC43 de coronavirus humains (HCoV). Un test immuno alternative indirecte (IPA) est décrite ici pour la détection et le titrage de ces virus. Cellules sensibles sont inoculés avec des dilutions de série logarithmique des échantillons dans une plaque de 96 puits. Après une croissance virale, la détection virale par l'IPA obtient le titre de virus infectieux, exprimé par la «dose infectieuse de culture tissulaire» (DICT50). Cela représente la dilution d'un échantillon contenant le virus au cours de laquelle la moitié d'une série de puits de laboratoire contiennent réplication virale. Cette technique est une méthode fiable pour le titrage du HCoV dans des échantillons biologiques (cellules, tissus ou les liquides).

Protocol

Le protocole texte complet de cette approche expérimentale est disponible en protocoles Springer .

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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