title: A neutralization assay for respiratory syncytial virus using a quantitative PCR-based endpoint assessment.
abstract: BACKGROUND
  Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting.
  
  
  METHODS
  Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies.
  
  
  RESULTS
  We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law).
  
  
  CONCLUSIONS
  This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.
authors.affiliation: Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA.
authors.forename: Jan C
authors.initials: JC
authors.lastname: Varada
authors.forename: Belete
authors.initials: B
authors.lastname: Teferedegne
authors.forename: R Lynne
authors.initials: RL
authors.lastname: Crim
authors.forename: Thembi
authors.initials: T
authors.lastname: Mdluli
authors.forename: Susette
authors.initials: S
authors.lastname: Audet
authors.forename: Keith
authors.initials: K
authors.lastname: Peden
authors.forename: Judy
authors.initials: J
authors.lastname: Beeler
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Murata
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D006801:Humans; D009500:Neutralization Tests / Q000379:methods*; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018113:Respiratory Syncytial Virus, Human / Q000276:immunology*; D019562:Viral Load / Q000379:methods*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral