免疫ペルオキシダーゼアッセイを用いたヒトコロナウイルスの滴定

Published 4/28/2008
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Biology
 

ERRATUM NOTICE

Summary

このビデオでは、我々は、免疫アッセイとして知られている酵素抗原の検出技術を使用して、ウイルスの検出とタイターのための代替方法を示しています。ここで、我々は、あなたのウイルスのサンプルを収集する方法を示して、テスト用のセルを準備し、最終的にウイルス力価を決定するために希釈系列を用いて免疫アッセイになります。

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

感染性ウイルス力価の計算は、ウイルス学者のための基本と本質的な実験的なアプローチを表しています。古典的なプラークアッセイは、ヒトコロナウイルスの株229EとOC43(HCOV)の場合がそうですが、重要な細胞変性効果を、発生しないウイルスには使用できません。別の間接免疫ペルオキシダーゼアッセイ(IPA)がここにこれらのウイルスの検出と滴定のために説明されています。感受性細胞を96ウェルプレート中のサンプルのシリアル対数希釈で接種されています。ウイルス増殖の後、IPAによるウイルスの検出は、"組織培養感染量"(TCID50)として表される感染性ウイルスの力価を、得られます。これは、複製ウイルスが含まれている実験室の井戸の一連のどの半分でウイルス含有試料の希釈を表しています。この手法は、生体試料中のHCOVの滴定(細胞、組織または体液)のための信頼性の高い方法です。

Protocol

この実験的なアプローチのための完全なテキストのプロトコルがで可能ですシュプリンガープロトコル

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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