Immunoperoksidaz Testi İnsan coronavirüsleri Titrasyon

Published 4/28/2008
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Biology
 

ERRATUM NOTICE

Summary

Bu video, immunoperoksidaz tahlil olarak bilinen bir enzimatik antijen algılama tekniği kullanılarak virüslerin tespit edilmesi ve titering için alternatif bir yöntem gösteriyor. Burada, biz, nasıl virüs örnekleri toplamak için size göstermek için test hücreleri hazırlamak ve viral titresi belirlemek için seri dilüsyonları kullanarak nihayet immunoperoksidaz tahlil olacak.

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

Bulaşıcı viral titreleri Hesaplama virologlar için bir temel ve temel deneysel bir yaklaşımı temsil ediyor. Klasik plak Testler insan coronavirüs (HCoV) suşları 229E ve OC43 durumda önemli sitopatik etkileri, yol açmazlar virüsler için kullanılamaz. Bu virüslerin saptanması ve titrasyonu için bir alternatif dolaylı immunoperoksidaz assay (IPA) burada açıklanmıştır. Duyarlı hücreler 96 plaka örnekleri seri logaritmik dilüsyonları inoküle. IPA virüs tespiti viral büyüme sonra, bulaşıcı bir virüs titresi, "Doku Kültürü Bulaşıcı Doz" (TCID50) olarak ifade verir. Bu, bir dizi laboratuar kuyuların yarısının kopyalayan virüs içeren bir virüs içeren bir örnek seyreltme temsil eder. Bu teknik, biyolojik örnekler HCoV titrasyonu (hücre, doku veya sıvıları) için güvenilir bir yöntem.

Protocol

Bu deneysel bir yaklaşım için tam metin protokolü mevcuttur Springer Protokolleri .

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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