免疫斑块试验（流感病毒）

Longping V.  Tse; Yueting  Zhang; Gary R.  Whittaker

doi:10.21769/BioProtoc.959

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Peer-reviewed

Immunoplaque Assay (Influenza Virus)

免疫斑块试验（流感病毒）

Longping V. Tse email

YZ Yueting Zhang

GW Gary R. Whittaker email

发布: 2013年11月05日第3卷第21期 DOI: 10.21769/BioProtoc.959 浏览次数: 24904

评审: Anonymous reviewer(s)

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The authors used this protocol in:

Cover of Journal of Virology, featuring study using the protocol.

Jun 2013

实验方案合集

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Abstract

Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification assays such as 50% tissue culture infectious assay (TCID₅₀) and genetic material quantification by Q-PCR provide a different way of viral quantification with their own shortcoming. In here, we modified the fluorescent focus assay and developed an antibody-based immunoplaque assay which provides a reliable and reproducible viral quantification independent of CPE. Our assay not only allows accurate determination of viral titer, but also provides information on viral kinetics, genetic stability and purity of the virus population.

Keywords: Influenza (禽流感)

Plaque Assay (试验板)

Quantification (量化的)

Materials and Reagents

MDCK cells
Please note that there are multiple variants of MDCK on American Type Culture Collection (ATCC). This protocol has been tested for MDCK (CCL-34), MDCK.1 (CRL-2935) and MDCK.2 (CRL-2936)
Recombinant A/California/04/2009 virus produced from 12 plasmids reverse genetic system (Generous gift from Professor Toru Takimoto from University of Rochester, New York, USA) (Bussey et al., 2010)
Trypsin/EDTA (Mediatech, Cellgro^®, catalog number: 25-053-CI )
SeaPlaque Agarose (Lonza, catalog number: 50100 )
TPCK-Trypsin (Thermo Fisher Scientific, catalog number: 20233 )
PBS without Ca²⁺ and Mg²⁺ (Mediatech, Cellgro^®, catalog number: 21-030-CV )
DPBS with Ca²⁺ and Mg²⁺ (Mediatech, Cellgro^®, catalog number: 21-040-CV )
10% formalin (Sigma-Aldrich, catalog number: HT501128-4L )
TritonX-100 (Sigma-Aldrich, catalog number: T9284 )
5% goat serum in DPBS (Gibco, catalog number: 16210-072 )
Mouse anti-NP antibody from hybridoma H16-L10-4R5 (ATCC, catalog number: HB-65 )
Alkaline phosphatase conjugated Donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories, catalog number: 715-056-150 )
Alexa Fluor 488 conjugated Goat anti-mouse antibody (Invitrogen, catalog number: A11001 )
BCIP/NBT substrate (Vector Laboratories, catalog number: SK-5400 )
DMEM (Mediatech, Cellgro^®, catalog number: 10-017-CV )
DMEM powder (Mediatech, Cellgro^®, catalog number: 50-003-PB )
Heat-inactivated fetal bovine serum (Mediatech, Cellgro^®, catalog number: 26140079 )
HEPES (Mediatech, Cellgro^®, catalog number: 25-060-CI )
Penicillin/Streptomycin (Mediatech, Cellgro^®, 30-002-CI )
7.5% BSA Fraction V (Gibco, catalog number: 15260 )
RPMI 1640 powder (Mediatech, Cellgro^®, catalog number: 50-020-PB )
Tris base (US Biological, catalog number: T8600 )
Sodium chloride (NaCl)
Magnesium chloride (MgCl₂)
Sodium hydroxide (NaOH)
Complete DMEM (cDMEM) (see Recipes)
2x DMEM (see Recipes)
RPMI (see Recipes)
0.5% Triton X-100 in DPBS (see Recipes)
Alkaline phosphatase (AP) reaction buffer (see Recipes)