Immunoperoxidase 분석을 사용하여 인간 Coronaviruses의 적정

Published 4/28/2008
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Biology
 

ERRATUM NOTICE

Summary

이 비디오에서 우리는 immunoperoxidase 분석으로 알려진 효소 항원 검출 기법을 사용하여 바이러스 감지 및 titering위한 대체 방법을 보여줍니다. 여기, 우리는 여러분의 바이러스 샘플을 수집하는 방법을 보여 테스트 세포를 준비하고, 바이러스 titer를 결정하는 일련 dilutions를 사용하여 최종 immunoperoxidase 분석됩니다.

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

전염성 바이러스 titers의 계산 virologists위한 기본적인 필수 실험 방법을 나타냅니다. 고전 플라크의 assays는 종자 229E 인간 coronavirus (HCoV)의 OC43의 경우는 상당한 cytopathic 효과를 발생하지 사용할 수있는 최신 바이러스 예방 프로그램으로 사용할 수 없습니다. 다른 간접 immunoperoxidase 분석 (IPA)는 본 이들 바이러스의 감지 및 적정에 대해 설명되어 있습니다. 민감한 세포는 96 - 웰 플레이트에 샘플 시리얼 로그 dilutions와 주사를하고 있습니다. 바이러스성 성장 후, I​​PA의 바이러스 검출은 "조직 문화 전염성 선량"(TCID50)로 표현 전염성 바이러스 titer를 산출. 이것은 실험실 우물 일련의 절반이 복제 바이러스를 포함하는에서 바이러스가 포함된 시료의 희석을 나타냅니다. 이 기술은 생물 학적 샘플에서 HCoV의 적정 (세포, 조직 또는 체액)에 대한 안정적인 방법입니다.

Protocol

이 실험 방법에 대한 전체 텍스트 프로토콜에서 사용할 수 있습니다 스프링거 프로토콜 .

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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