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Real-time quantitative PCR-based serum neutralization test for detection and titration of neutralizing antibodies to chicken anemia virus

Authors :
Vicky L. van Santen
Kenneth S. Macklin
Kellye S. Joiner
R. A. Norton
Bernhard Kaltenboeck
Source :
Journal of Virological Methods. 115:123-135
Publication Year :
2004
Publisher :
Elsevier BV, 2004.

Abstract

Detection and titration of chicken anemia virus (CAV)-neutralizing antibodies has relied on tedious, time-consuming passaging of infected cells, or subjective recognition of cytopathic effect in individual cells, because CAV replicates in culture only in lymphoblastoid cell lines, and thus generates no plaques. This paper describes a rapid method, in which CAV genomes in infected cells are quantitated by qPCR 3-4 days postinfection (p.i.), without passaging cells. Three sera, weakly positive with a commercial CAV ELISA kit, from broiler chickens immunized with a commercial CAV vaccine, were used to develop the assay. Virus neutralization titers of these sera were determined using two different CAV-susceptible cell lines (MDCC-MSB1 and MDCC-CU147) by the conventional method of passaging cells infected with 10,000 TCID(50) CAV per well, and by qPCR-based methods using cells infected with 100 or 10,000 TCID(50) per well in 24-well or 96-well plates. The method was also adapted to conventional PCR. The positive sera exhibited virus neutralization activity at dilutions ranging from 1:10 to 1:320 by the various assays. Although virus neutralization titers differed somewhat depending on the assay conditions used, the relative order of the titers of the three positive sera was the same for all assays. The qPCR-based assays are as sensitive and more rapid for detection of neutralizing antibody than the conventional assay based on passaging infected cells, and more sensitive for detection of low-level CAV antibodies than a commercial blocking ELISA.

Details

ISSN :
01660934
Volume :
115
Database :
OpenAIRE
Journal :
Journal of Virological Methods
Accession number :
edsair.doi.dedup.....727a15938a258921e5a1c31822fae07f
Full Text :
https://doi.org/10.1016/j.jviromet.2003.09.022