Volume 88, Issue 5
  • ISSN: 0002-9637
  • E-ISSN: 1476-1645



A dengue plaque reduction neutralization test (PRNT) to measure dengue serotype–specific neutralizing antibodies for all four virus serotypes was developed, optimized, and validated in accordance with guidelines for validation of bioanalytical test methods using human serum samples from dengue-infected persons and persons receiving a dengue vaccine candidate. Production and characterization of dengue challenge viruses used in the assay was standardized. Once virus stocks were characterized, the dengue PRNT for each of the four serotypes was optimized according to a factorial design of experiments approach for critical test parameters, including days of cell seeding before testing, percentage of overlay carboxymethylcellulose medium, and days of incubation post-infection to generate a robust assay. The PRNT was then validated and demonstrated to be suitable to detect and measure dengue serotype-specific neutralizing antibodies in human serum samples with acceptable intra-assay and inter-assay precision, accuracy/dilutability, specificity, and with a lower limit of quantitation of 10.

[open-access] This is an Open Access article distributed under the terms of the American Society of Tropical Medicine and Hygiene's Re-use License which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Article metrics loading...

The graphs shown below represent data from March 2017
Loading full text...

Full text loading...



  1. Gubler DJ, , 2002. Epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the 21st century. Trends Microbiol 10: 100103.[Crossref] [Google Scholar]
  2. Lindenbach BD, Rice CM, , 2003. Molecular biology of flaviviruses. Adv Virus Res 59: 2361.[Crossref] [Google Scholar]
  3. World Health Organization Regional Office for South-East Asia, 2011. Comprehensive Guidelines for Prevention and Control of Dengue and Dengue Haemorrhagic Fever. Available at: http://www.searo.who.int/LinkFiles/Dengue_DHF_prevention&control_guidelines_rev.pdf. [Google Scholar]
  4. Thomas SJ, Endy TP, , 2011. Vaccines for the prevention of dengue: development update. Hum Vaccin 7: 674684.[Crossref] [Google Scholar]
  5. Murphy BR, Whitehead SS, , 2011. Immune response to dengue virus and prospects for a vaccine. Annu Rev Immunol 29: 587619.[Crossref] [Google Scholar]
  6. Clarke DH, Casals J, , 1958. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg 7: 561573. [Google Scholar]
  7. Centers for Disease Control, 1981. A Guide to the Performance of the Standardized Diagnostic Complement Fixation Method and Adaptation to Micro Test. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control. [Google Scholar]
  8. Casey HL, , 1965. Standardized diagnostic complement fixation method and adaptation to micro test. Public Health Monogr 74: 134. [Google Scholar]
  9. Chungue E, Marche G, Plichart R, Boutin JP, Roux J, , 1989. Comparison of immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA) and haemagglutination inhibition (HI) test for the detection of dengue antibodies. Prevalence of dengue IgG-ELISA antibodies in Tahiti. Trans R Soc Trop Med Hyg 83: 708711.[Crossref] [Google Scholar]
  10. Miagostovich MP, Nogueira RM, dos Santos FB, Schatzmayr HG, Araujo ES, Vorndam V, , 1999. Evaluation of an IgG enzyme-linked immunosorbent assay for dengue diagnosis. J Clin Virol 14: 183189.[Crossref] [Google Scholar]
  11. Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S, Puttisri P, Hoke CH, , 1989. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg 40: 418427. [Google Scholar]
  12. Thomas SJ, Nisalak A, Anderson KB, Libraty DH, Kalayanarooj S, Vaughn DW, Putnak R, Gibbons RV, Jarman R, Endy TP, , 2009. Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infections: how alterations in assay conditions impact performance. Am J Trop Med Hyg 81: 825833.[Crossref] [Google Scholar]
  13. Russell PK, Nisalak A, Sukhavachana P, Vivona S, , 1967. A plaque reduction test for dengue virus neutralizing antibodies. J Immunol 99: 285290. [Google Scholar]
  14. Putnak JR, de la Barrera R, Burgess T, Pardo J, Dessy F, Gheysen D, Lobet Y, Green S, Endy TP, Thomas SJ, Eckels KH, Innis BL, Sun W, , 2008. Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies. Am J Trop Med Hyg 79: 115122. [Google Scholar]
  15. Roehrig JT, Hombach J, Barrett AD, , 2008. Guidelines for plaque-reduction neutralization testing of human antibodies to dengue viruses. Viral Immunol 21: 123132.[Crossref] [Google Scholar]
  16. Yoksan S, Tubthong K, Kanitwithayanun W, Jirakanjanakit N, , 2009. Laboratory assays and field dengue vaccine evaluation at Ratchaburi province, Thailand: a preliminary result. J Clin Virol 46 (Suppl 2): S13S15.[Crossref] [Google Scholar]
  17. Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N, , 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 22: 250254. [Google Scholar]
  18. World Health Organization, 2007. Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses. WHO/IVB/07.07. Available at: http://whqlibdoc.who.int/hq/2007/who_ivb_07.07_eng.pdf. [Google Scholar]
  19. Guy B, Barrere B, Malinowski C, Saville M, Teyssou R, Lang J, , 2011. From research to phase III: preclinical, industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine. Vaccine 29: 72297241.[Crossref] [Google Scholar]
  20. Lang J, , 2009. Recent progress on Sanofi Pasteur's dengue vaccine candidate. J Clin Virol 46 (Suppl 2): S20S24.[Crossref] [Google Scholar]
  21. Edelman R, Wasserman SS, Bodison SA, Putnak RJ, Eckels KH, Tang D, Kanesa-Thasan N, Vaughn DW, Innis BL, Sun W, , 2003. Phase I trial of 16 formulations of a tetravalent live-attenuated dengue vaccine. Am J Trop Med Hyg 69 (Suppl 6): 4860. [Google Scholar]
  22. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chambonneau L, Saluzzo JF, Bhamarapravati N, , 2004. Safety and immunogenicity of a three dose regimen of two tetravalent live-attenuated dengue vaccines in five- to twelve-year-old Thai children. Pediatr Infect Dis J 23: 99109.[Crossref] [Google Scholar]
  23. Sabchareon A, Lang J, Chanthavanich P, Yoksan S, Forrat R, Attanath P, Sirivichayakul C, Pengsaa K, Pojjaroen-Anant C, Chokejindachai W, Jagsudee A, Saluzzo JF, Bhamarapravati N, , 2002. Safety and immunogenicity of tetravalent live-attenuated dengue vaccines in Thai adult volunteers: role of serotype concentration, ratio, and multiple doses. Am J Trop Med Hyg 66: 264272. [Google Scholar]
  24. Simasathien S, Thomas SJ, Watanaveeradej V, Nisalak A, Barberousse C, Innis BL, Sun W, Putnak JR, Eckels KH, Hutagalung Y, Gibbons RV, Zhang C, De La Barrera R, Jarman RG, Chawachalasai W, Mammen MP, Jr, 2008. Safety and immunogenicity of a tetravalent live-attenuated dengue vaccine in flavivirus naive children. Am J Trop Med Hyg 78: 426433. [Google Scholar]
  25. International Conference on Harmonization, 1995. Guidance for Industry: Validation of Analytical Procedures. Document Q2A. Available at: http://www.fda.gov/cder/guidance/ichq2a.pdf. [Google Scholar]
  26. International Conference on Harmonization, 1996. Guidance for Industry: Validation of Analytical Procedures. Document Q2B. Available at: http://www.fda.gov/cder/guidance/ichq2b.pdf. [Google Scholar]
  27. Food and Drug Administration, 2001. Guidance for Industry: Bioanalytical Method Validation. Available at: http://www.fda.gov/cder/guidance/4252fnl.pdf. [Google Scholar]
  28. Guy B, Chanthavanich P, Gimenez S, Sirivichayakul C, Sabchareon A, Begue S, Yoksan S, Luxemburger C, Lang J, , 2004. Evaluation by flow cytometry of antibody-dependent enhancement (ADE) of dengue infection by sera from Thai children immunized with a live-attenuated tetravalent dengue vaccine. Vaccine 22: 35633574.[Crossref] [Google Scholar]
  29. Mota J, Acosta M, Argotte R, Figueroa R, Mendez A, Ramos C, , 2005. Induction of protective antibodies against dengue virus by tetravalent DNA immunization of mice with domain III of the envelope protein. Vaccine 23: 34693476.[Crossref] [Google Scholar]
  30. Guirakhoo F, Arroyo J, Pugachev KV, Miller C, Zhang ZX, Weltzin R, Georgakopoulos K, Catalan J, Ocran S, Soike K, Ratterree M, Monath TP, , 2001. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75: 72907304.[Crossref] [Google Scholar]
  31. Guirakhoo F, Weltzin R, Chambers TJ, Zhang ZX, Soike K, Ratterree M, Arroyo J, Georgakopoulos K, Catalan J, Monath TP, , 2000. Recombinant chimeric yellow fever-dengue type 2 virus is immunogenic and protective in nonhuman primates. J Virol 74: 54775485.[Crossref] [Google Scholar]
  32. Sithisarn P, Suksanpaisan L, Thepparit C, Smith DR, , 2003. Behavior of the dengue virus in solution. J Med Virol 71: 532539.[Crossref] [Google Scholar]
  33. Morens DM, Halstead SB, Larsen LK, , 1985. Comparison of dengue virus plaque reduction neutralization by macro and “semi-micro” methods in LLC-MK2 cells. Microbiol Immunol 29: 11971205.[Crossref] [Google Scholar]
  34. Matsumura T, Stollar V, Schlesinger RW, , 1972. Effects of ionic strength on the release of dengue virus from Vero cells. J Gen Virol 17: 343347.[Crossref] [Google Scholar]
  35. Brandler S, Brown N, Ermak TH, Mitchell F, Parsons M, Zhang Z, Lang J, Monath TP, Guirakhoo F, , 2005. Replication of chimeric yellow fever virus-dengue serotype 1–4 virus vaccine strains in dendritic and hepatic cells. Am J Trop Med Hyg 72: 7481. [Google Scholar]
  36. Gupta CK, Leszczynski J, Gupta RK, Siber GR, , 1996. Stabilization of respiratory syncytial virus (RSV) against thermal inactivation and freeze-thaw cycles for development and control of RSV vaccines and immune globulin. Vaccine 14: 14171420.[Crossref] [Google Scholar]
  37. Hunter-Cevera JC, Belt A, , 1996. Maintaining Cultures for Biotechnology and Industry. San Diego, CA: Academic Press. [Google Scholar]
  38. Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B, Halstead SB, , 1984. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol 120: 653569.[Crossref] [Google Scholar]
  39. Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, , 2002. Spatial and temporal circulation of dengue virus serotypes: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156: 5259.[Crossref] [Google Scholar]
  40. Moi ML, Lim CK, Kotaki A, Takasaki T, Kurane I, , 2010. Discrepancy in dengue virus neutralizing antibody titers between plaque reduction neutralizing tests with Fcgamma receptor (FcgammaR)-negative and FcgammaR-expressing BHK-21 cells. Clin Vaccine Immunol 17: 402407.[Crossref] [Google Scholar]
  41. Beaty BJ, Calisher CH, Shope RE, Leanette EH, Schmidt NJ, , 1995. Arboviruses. In: , eds. Diagnostic Procedures for Viral, Rickettsial and Chyamydial Infections. Seventh edition. American Public Health Association, 189212. [Google Scholar]
  42. Martin NC, Pardo J, Simmons M, Tjaden JA, Widjaja S, Marovich MA, , 2006. An immunocytometric assay based on dengue infection via DC-SIGN permits rapid measurement of anti-dengue neutralizing antibodies. J Virol Methods 134: 7485.[Crossref] [Google Scholar]
  43. Kraus AA, Messer W, Haymore LB, de Silva AM, , 2007. Comparison of plaque- and flow cytometry-based methods for measuring dengue virus neutralization. J Clin Microbiol 45: 37773780.[Crossref] [Google Scholar]
  44. Vorndam V, Beltran M, , 2002. Enzyme-linked immunosorbent assay-format microneutralization test for dengue viruses. Am J Trop Med Hyg 66: 208212. [Google Scholar]
  45. Ting SH, See E, Tan HC, Lee MA, Ooi EE, , 2001. Development of a simplified assay for the detection of neutralizing antibodies to Japanese encephalitis virus. J Virol Methods 93: 4347.[Crossref] [Google Scholar]
  46. Jirakanjanakit N, Sanohsomneing T, Yoksan S, Bhamarapravati N, , 1997. The micro-focus reduction neutralization test for determining dengue and Japanese encephalitis neutralizing antibodies in volunteers vaccinated against dengue. Trans R Soc Trop Med Hyg 91: 614617.[Crossref] [Google Scholar]
  47. Thacker WL, Lewis VJ, Baer GM, Sather GE, , 1978. A rapid fluorescent focus-inhibition test for determining dengue neutralizing antibody and for identifying prototype dengue viruses. Can J Microbiol 24: 15531556.[Crossref] [Google Scholar]

Data & Media loading...

  • Received : 30 Jul 2012
  • Accepted : 25 Jan 2013
  • Published online : 01 May 2013

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error