Waiting
Login processing...

Trial ends in Request Full Access Tell Your Colleague About Jove
Click here for the English version

Biology

Titolazione del coronavirus umani utilizzando un test immunoperossidasi

doi: 10.3791/751 Published: April 28, 2008

ERRATUM NOTICE

Summary

In questo video, dimostriamo un metodo alternativo per il rilevamento e la titolazione di virus utilizzando una tecnica di rilevazione enzimatica antigene conosciuto come un saggio immunoperossidasi. Qui, vi mostreremo come raccogliere i campioni virali, preparare le cellule per il test, e infine il saggio immunoperossidasi utilizzando diluizioni seriali per determinare il titolo virale.

Abstract

Calcolo del infettive titoli virale rappresenta un approccio di base e sperimentali indispensabile per virologi. Saggi di placca classica non può essere utilizzato per i virus che non causino un rilevante effetto citopatico, che è il caso per i ceppi OC43 e 229E di coronavirus umani (HCoV). Un test alternativo immunoperossidasi indiretti (IPA) è qui descritto per la rilevazione e la titolazione di questi virus. Cellule sensibili vengono inoculate con diluizioni seriali logaritmica di campioni in una piastra da 96 pozzetti. Dopo una crescita virale, il rilevamento virale da IPA fornisce il titolo infettivo dei virus, espressa come "Dose Tissue Culture Infettive" (TCID50). Questo rappresenta la diluizione di un virus contenente campione a cui la metà di una serie di pozzi di laboratorio contengono virus replicante. Questa tecnica è un metodo affidabile per la titolazione di HCoV in campioni biologici (cellule, tessuti o fluidi).

Protocol

Il protocollo testo completo di questo approccio sperimentale è disponibile in protocolli di 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).

Titolazione del coronavirus umani utilizzando un test immunoperossidasi
Play Video
PDF DOI

Cite this Article

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).More

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).

Less
Copy Citation Download Citation Reprints and Permissions
View Video

Get cutting-edge science videos from JoVE sent straight to your inbox every month.

Waiting X
Simple Hit Counter