Titulação de coronavírus humanos Usando um ensaio de imunoperoxidase

Published 4/28/2008
3 Comments
  CITE THIS  SHARE 
Biology
 

ERRATUM NOTICE

Summary

Neste vídeo, demonstramos um método alternativo para a detecção e titulação de vírus usando uma técnica de detecção de antígeno enzimático conhecido como um ensaio de imunoperoxidase. Aqui, vamos mostrar-lhe como coletar as amostras virais, preparar as células para testes, e, finalmente, o ensaio de imunoperoxidase utilizando diluições de série para determinar o título viral.

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).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

Cálculo de doenças infecciosas virais títulos representa uma abordagem básica e essencial experimental para virologistas. Ensaios de placa clássico não pode ser usado para vírus que não causam efeitos significativos citopático, o que é o caso de cepas 229E e OC43 de coronavírus humano (HCoV). Um ensaio de imunoperoxidase indireta alternativa (IPA) é aqui descrita para a detecção e titulação desses vírus. Células susceptíveis são inoculadas com diluições de série logarítmica de amostras em uma placa de 96 poços. Após o crescimento viral, a detecção viral por IPA produz o título de vírus infecciosos, expresso como "Cultura de Tecidos Dose Infecciosa" (TCID50). Isto representa a diluição de uma amostra de vírus contendo em que metade de uma série de poços de laboratório contêm vírus replicantes. Esta técnica é um método confiável para a titulação de HCoV em amostras biológicas (células, tecidos ou fluidos).

Protocol

O protocolo de texto completo para essa abordagem experimental está disponível em Protocolos 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

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Video Stats