Abstract
A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID50). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.
Highlights
Influenza serological studies are performed frequently in the context of clinical diagnosis for identification of human infection, global surveillance for circulating and emerging virus strains, virus antigenic characterization for seasonal vaccine-strain selection, and immunogenicity evaluation for vaccine licensure [1]
Lysates from MDCKLondon cells grown in 96-well culture plates were prepared by washing the cells once with PBS and adding 100 mL of Sample Preparation Reagent (SPR) per well
In order to generate a quantification standard for quantitative reverse transcription PCR (qRT-PCR), total RNA was purified from Madin-Darby canine kidney (MDCK)-London cells infected with the influenza virus strain A/PR/8/34 and a dilution series was prepared using SPR lysate from uninfected MDCKLondon cells as the diluent
Summary
Influenza serological studies are performed frequently in the context of clinical diagnosis for identification of human infection, global surveillance for circulating and emerging virus strains, virus antigenic characterization for seasonal vaccine-strain selection, and immunogenicity evaluation for vaccine licensure [1]. The hemagglutination-inhibition (HI) assay presently remains in wide use, in part owing to its relative simplicity and the existence of clinical data in support of establishing a serological correlate of protection against infection (a serum HI titer of at least 40 is generally considered to provide meaningful protection) [2,3]. MN assays appear to exhibit higher sensitivity for the detection of low-titer seroconversions [9]. The higher sensitivity of MN assays may be useful in the assessment of human antibodies against avian influenza viruses [10]. Despite their advantages, MN assays appear to be associated with significantly greater inter- and intralaboratory variability compared with the HI assay [11,12,13]
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