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Journal of Clinical Microbiology, July 2003, p. 3154-3157, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3154-3157.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Improved Assay To Detect Neutralizing Antibody following Vaccination with Diluted or Undiluted Vaccinia (Dryvax) Vaccine

Saint Louis University Vaccine Treatment and Evaluation Unit, St. Louis, Missouri,¹ The EMMES Corporation, Rockville, Maryland²

Received 24 September 2002/ Returned for modification 3 March 2003/ Accepted 1 May 2003

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.

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