title: Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies.
abstract: Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay-based microneutralization (MN) assay, and a fluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.
authors.affiliation: Division of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA. robert.putnak@na.amedd.army.mil
authors.forename: J Robert
authors.initials: JR
authors.lastname: Putnak
authors.forename: Rafael
authors.initials: R
authors.lastname: de la Barrera
authors.forename: Timothy
authors.initials: T
authors.lastname: Burgess
authors.forename: Jorge
authors.initials: J
authors.lastname: Pardo
authors.forename: Francis
authors.initials: F
authors.lastname: Dessy
authors.forename: Dirk
authors.initials: D
authors.lastname: Gheysen
authors.forename: Yves
authors.initials: Y
authors.lastname: Lobet
authors.forename: Sharone
authors.initials: S
authors.lastname: Green
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Endy
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Thomas
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Eckels
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Innis
authors.forename: Wellington
authors.initials: W
authors.lastname: Sun
mesh_terms: D000914:Antibodies, Viral / Q000737:chemistry / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D003716:Dengue Virus / Q000145:classification / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D005189:False Positive Reactions; D009500:Neutralization Tests / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral