The variation in PRNT titers was demonstrated not only to be impacted by manipulating a single condition but also by interaction between two or more conditions. The effect of viral passage on NAb titers was significant but not standard across all DENV types. are characterized mainly because small, enveloped viruses containing a single positive strand of ribonucleic acid (RNA) and are members of the family assay using plaque reduction to measure DENV neutralizing antibody and DENV recognition was developed in 1967 by Russell and Nisalak.11,12 The Russell and UNC-2025 Nisalak assay became known as the plaque reduction neutralization test (PRNT) and used prototype dengue seed viruses, monkey anti-sera controls, LLC-MK2 cell lines, and an agar overlay press with neutral red staining. A probit analysis was used to determine the serum titer required to reduce dengue viral plaques by 50% (PRNT50) compared with control. The PRNT launched a method of measuring DENV type-specific neutralizing antibodies and offers remained the standard assay. Variations of the Russell PRNT were subsequently introduced using a variety of cell lines and methods: 1) a micro-metabolic inhibition test and a microculture plaque-reduction test using BHK-21 (baby hamster kidney cells) and LLC-MK2 cells lines, respectively; 2) microplate cultures using BHK-21 cells, a focus reduction method using peroxidase-anti-peroxidase staining of BHK-21 cells; 3) a semi-micro method in LLC-MK2 cells using a 70% plaque reduction criteria; and 4) a simplified PRNT assay using BHK-21 cells.13C17 Today a wide variety of dengue PRNT assays are being used by dengue vaccine designers, academic UNC-2025 study, and public health laboratories. The PRNT is being used to define the immunogenicity of dengue vaccine candidates, support dengue seroepidemiologic studies, and support pathogenesis studies of severe dengue illness.18C29 Despite its widespread use, neither the PRNT nor the required critical reagents (e.g., cell Rabbit Polyclonal to Collagen VI alpha2 collection, viral strains, passage, complement) have been standardized nor harmonized between laboratories. Recommendations within the conduct of the PRNT have recently been published by the World Health Corporation (WHO) Initiative for Vaccine Study of the Division of Immunization, Vaccines and Biologicals with support from your Expenses and Melinda Gates Basis Pediatric Dengue Vaccine Initiative ( We conducted a series of experiments to define the variability in anti-dengue virus PRNT effects using different cell lines, virus preparations, and the presence or absence of complement. Our study demonstrated that changes of these conditions had significant effects within the PRNT titers measured in a given serum sample. Significant associations were observed between particular testing conditions and raises and decreases in titers from UNC-2025 different checks on the same serum sample. These findings underscore the need to harmonize assay methods, testing conditions, and important reagents if inter-laboratory assessment of PRNT results is desired. Materials and Methods Standardized sera panel A standardized sera panel was UNC-2025 used to test the performance of the PRNT under a variety of test conditions. The panel was put together from blood samples collected as part of a hospital-based study evaluating children with suspected dengue admitted to the Queen Sirikit National Institute of Child Health (QSNICH) located in Bangkok, Thailand.31 The study was approved by the Thai Ministry of Health, QSNICH, University or college of Massachusetts Medical School, and U.S. Army ethical evaluate committees. All volunteers were enrolled following an informed consent process with parent(s) and written documentation of the same. Sera were characterized for the presence of dengue antibody by dengue enzyme immunoassay (EIA), hemagglutination inhibition (HAI), mosquito inoculation with viral isolation, and DENV recognition by a typing enzyme immunoassay.32C35 A diagnosis of dengue and clinical characterization were guided by founded criteria (WHO, monograph on Dengue/Dengue Hemorrhagic Fever [1997]) applied by a medical monitor, as previously described and outlined below.36 Paired sera from 18 individuals were used in all neutralization assays (Table 1) testing all conditions (Number 1). Acute samples were acquired between 8 and 11 days after hospital admission and late convalescent samples were obtained 354C380 days after admission; one convalescent sample was acquired 177 days after hospital admission. The panel consisted of 6 main dengue infections (3 DENV-1, 1 DENV-2, and 2 DENV-3) and 12 secondary dengue infections (3 DENV-1, 3 DENV-2, 3 DENV-3, and 3 DENV-4). Open in a separate window Number 1.