A pseudo-plaque reduction neutralization test was applied to CCHFV-neutralizing antibody detection in a 96-well microplate scale

A pseudo-plaque reduction neutralization test was applied to CCHFV-neutralizing antibody detection in a 96-well microplate scale. of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. Methods Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was decided and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. Results Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. Conclusion The pseudo-plaque reduction neutralization test described in this study is usually a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture. family Bunyaviridae, which are enveloped viruses containing tripartite, unfavorable polarity, single-stranded RNA [1,2]. Crimean-Congo hemorrhagic fever, a severe viral human disease, is characterized by sudden onset of fever, headache, abdominal pain, nausea, vomiting, extensive ecchymoses, bleeding, and hepatic dysfunction with fatality rates up to 30% [3,4]. The virus is transmitted to humans by the bite of infected ticks, by squashed ticks, or by exposure to the tissue or blood of infected livestock [5,6]. Crimean-Congo hemorrhagic fever virus can spread from person to person through contact with the tissue or blood of CCHF patients. It is one of the rare hemorrhagic fever viruses capable of inducing nosocomial outbreaks which may result in a more severe illness with a higher mortality rate [7-10]. Crimean-Congo hemorrhagic fever is usually diagnosed genetically ML604086 by detection of viral RNA in acute-phase blood sample or serum [3,4,9-12]. Serological diagnosis relies on ML604086 detection of anti-CCHF specific IgM and IgG in enzyme-linked immunosorbent (ELISA) and immunofluorescence assays (IFA) from paired acute and convalescent specimens [13-17]. Ideally, the confirmation of CCHF contamination should be made by neutralization assay which is one of the most specific serological methods. Virus neutralization tests are usually based on the cytopathic effect (CPE) or the plaque-reduction neutralization test (PRNT) [18,19]. The CPE assay relies on the visual examination of the damage in magnified infected target cells. It is subjected to observer variation and it is difficult to make a quantitative determination of neutralizing activity based on the CPE. The PRNT has limitations for screening the large numbers of serum samples needed for epidemiological investigations. Neither CPE assay nor PRNT can be used to measure neutralization antibodies if the virus produces little or no CPE. A pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with ML604086 anti-CCHFV antibodies was Rat monoclonal to CD8.The 4AM43 monoclonal reacts with the mouse CD8 molecule which expressed on most thymocytes and mature T lymphocytes Ts / c sub-group cells.CD8 is an antigen co-recepter on T cells that interacts with MHC class I on antigen-presenting cells or epithelial cells.CD8 promotes T cells activation through its association with the TRC complex and protei tyrosine kinase lck used to measure neutralization antibody of CCHFV. The results obtained by PPRNT were compared with those of a fluorescence focus reduction neutralization test (FFRNT). Results CCHFV pseudo-plaque reduction neutralization assay Crimean-Congo hemorrhagic fever Turkey-Kelkit06 strain does not produce plaques. We have been able to titrate the virus by the recently developed pseudo-plaque assay (PPA) described by Mitchell et al. [20] with some modifications. A pseudo-plaque reduction neutralization test was applied to CCHFV-neutralizing antibody detection in a 96-well microplate scale. Crimean-Congo hemorrhagic fever from challenged serial dilutions.