Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR

• Published in: PloS one 2013-02, Vol.8 (2), p.e56023-e56023
• Main Authors: Teferedegne, Belete, Lewis, Jr, Andrew M, Peden, Keith, Murata, Haruhiko
• Format: Article
• Language: English
• Subjects: Analysis; Animals; Antisera; Assaying; Automation; Avian flu; Biology; Blood; Cell culture; Deoxyribonucleic acid; Dilution; DNA; Dogs; Endpoint Determination - methods; Enzyme-Linked Immunosorbent Assay; Experiments; Feasibility studies; Food; Green chemistry; Green development; Growth curves; Health aspects; Immunoglobulins; Infections; Influenza; Influenza A; Influenza A virus - genetics; Influenza A virus - physiology; Influenza vaccines; Influenza viruses; Kinetics; Laboratories; Madin Darby Canine Kidney Cells; Medicine; Neutralization; Neutralization Tests - methods; Pandemics; Primers; Reagents; Reverse Transcriptase Polymerase Chain Reaction - methods; Reverse transcription; Swine flu; Transcription (Genetics); Vaccination; Vaccines; Virus Replication - physiology; Viruses; West Nile virus
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0056023

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 TCID(50)). 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.