title: Improved assay to detect neutralizing antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine.
abstract: The assessment of immunogenicity of a diluted vaccinia vaccine for possible widespread use of a diluted vaccine in the event of a bioterrorist attack prompted us to focus on the development of a sensitive and specific plaque reduction neutralization (PRN) assay to assess the antibody response of volunteers to a vaccinia (Dryvax) vaccine. Two incubation times, 1 h or overnight (approximately 15 h), were explored for the neutralization step of the assay. In addition, serum samples were evaluated using both sonicated and nonsonicated virus in PRN assays with 1 and 15 h of incubation. The use of the overnight incubation method resulted in the detection of antibody in two vaccinated individuals who exhibited a take, i.e., a major reaction indicative of successive vaccination as defined by the Centers for Disease Control and Prevention, but did not have a fourfold increase in antibody to vaccinia virus by the 1-h-incubation method and increased the sensitivity from 94 to 100%. In addition to the increased sensitivity of the assay, we noted a significant increase (approximately 40-fold) in the PRN titer of serum samples tested with the 15-h-incubation method. The use of sonicated virus increased the reproducibility of the virus titers and PRN titers. Forty-two percent of the samples tested using sonicated virus had a PRN titer that was fourfold higher or greater than that of nonsonicated virus in the assay. A PRN titer that was threefold higher or greater was observed in more than half (58%) of the samples using sonicated virus. Therefore, the more sensitive, specific, and reproducible plaque neutralization assay for the detection of antibody to vaccinia virus is the method using a 15-h-incubation time and freshly sonicated vaccinia virus.
authors.affiliation: Saint Louis University Vaccine Treatment and Evaluation Unit, St. Louis, Missouri 63110, USA. newmanfk@slu.edu
authors.forename: Frances K
authors.initials: FK
authors.lastname: Newman
authors.forename: Sharon E
authors.initials: SE
authors.lastname: Frey
authors.forename: Tamara P
authors.initials: TP
authors.lastname: Blevins
authors.forename: Mahendra
authors.initials: M
authors.lastname: Mandava
authors.forename: Andres
authors.initials: A
authors.lastname: Bonifacio
authors.forename: Lihan
authors.initials: L
authors.lastname: Yan
authors.forename: Robert B
authors.initials: RB
authors.lastname: Belshe
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D006801:Humans; D009500:Neutralization Tests; D012680:Sensitivity and Specificity; D012899:Smallpox / Q000517:prevention & control*; D012900:Smallpox Vaccine / Q000008:administration & dosage* / Q000276:immunology; D013010:Sonication; D014611:Vaccination; D014616:Vaccinia virus / Q000276:immunology*; D010948:Viral Plaque Assay
chemical_list: D000914:Antibodies, Viral; D012900:Smallpox Vaccine