Skip to main content
Virologica Sinica logoLink to Virologica Sinica
. 2014 Dec 15;29(6):372–380. doi: 10.1007/s12250-014-3512-0

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Yi Huang 1, Youjie Zhu 1,2, Mengshi Yang 1, Zhenqing Zhang 1, Donglin Song 1, Zhiming Yuan 1,
PMCID: PMC8206289  PMID: 25547682

Abstract

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA’s ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

Keywords: Ebola virus, Marburg virus, recombinant nucleoproteins, indirect ELISA

References

  1. Barrette R W, Metwally S A, Rowland J M, Xu L, Zaki S R, Nichol S T, Rollin P E, Towner J S, Shieh W J, Batten B, Sealy T K, Carrillo C, Moran K E, Bracht A J, Mayr G A, Sirios-Cruz M, Catbagan D P, Lautner E A, Ksiazek T G, White W R, McIntosh M T. Discovery of swine as a host for the Reston ebolavirus. Science. 2009;325:204–206. doi: 10.1126/science.1172705. [DOI] [PubMed] [Google Scholar]
  2. Bharat T A, Noda T, Riches J D, Kraehling V, Kolesnikova L, Becker S, Kawaoka Y, Briggs J A. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci USA. 2012;109:4275–4280. doi: 10.1073/pnas.1120453109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Feldmann H, Jones S, Klenk H D, Schnittler H J. Ebola virus: from discovery to vaccine. Nat. Rev. Immunol. 2003;3:677–685. doi: 10.1038/nri1154. [DOI] [PubMed] [Google Scholar]
  4. Huang Y, Wei H, Wang Y, Shi Z, Raoul H, Yuan Z. Rapid detection of filoviruses by real-time TaqMan polymerase chain reaction assays. Virol Sin. 2012;27:273–277. doi: 10.1007/s12250-012-3252-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ikegami T, Niikura M, Saijo M, Miranda M E, Calaor A B, Hernandez M, Acosta L P, Manalo D L, Kurane I, Yoshikawa Y, Morikawa S. Antigen capture enzyme-linked immunosorbent assay for specific detection of Reston Ebola virus nucleoprotein. Clin Diagn Lab Immunol. 2003;10:552–557. doi: 10.1128/CDLI.10.4.552-557.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jaax N, Jahrling P, Geisbert T, Geisbert J, Steele K, McKee K, Nagley D, Johnson E, Jaax G, Peters C. Transmission of Ebolavirus (Zaire strain) to uninfected control monkeys in a biocontainment laboratory. Lancet. 1995;346:1669–1671. doi: 10.1016/S0140-6736(95)92841-3. [DOI] [PubMed] [Google Scholar]
  7. Ksiazek T G, West C P, Rollin P E, Jahrling P B, Peters C J. ELISA for the detection of antibodies to Ebola viruses. J Infect Dis. 1999;179:S192–S198. doi: 10.1086/514313. [DOI] [PubMed] [Google Scholar]
  8. Ksiazek T G, Rollin P E, Williams A J, Bressler D S, Martin M L, Swanepoel R, Burt F J, Leman P A, Khan A S, Rowe A K, Mukunu R, Sanchez A, Peters C J. Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigens, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995. J Infect Dis. 1999;179:S177–S189. doi: 10.1086/514321. [DOI] [PubMed] [Google Scholar]
  9. Kuhn J H, Bao Y M, Bavari S, Becker S, Bradfute S, Brister J R, Bukreyev A A, Cai Y, Chandran K, Davey R A, Dolnik O, Dye J M, Enterlein S, Gonzalez J P, Formenty P, Freiberg A N, Hensley L E, Honko A N, Ignatyev G M, Jahrling P B, Johnson K M, Klenk H D, Kobinger G, Lackemeyer M G, Leroy E M, Lever M S, Lofts L L, Mühlberger E, Netesov S V, Olinger G G, Palacios G, Patterson J L, Paweska J T, Pitt L, Radoshitzky S R, Ryabchikova E I, Saphire E O, Shestopalov A M, Smither S J, Sullivan N J, Swanepoel R, Takada A, Towner J S, van der Groen G, Volchkov V E, Wahl-Jensen V, Warren T K, Warfield K L, Weidmann M, Nichol S T. Virus nomenclature below the species level: a standardized nomenclature for laboratory animal-adapted strains and variants of viruses assigned to the family Filoviridae. Arch Virol. 2013;158:1425–1432. doi: 10.1007/s00705-012-1594-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Leroy E M, Kumulungui B, Pourrut X, Rouquet P, Hassanin A, Yaba P, De’licat A, Paweska J T, Gonzalez J P, Swanepoel R. Fruit bats as reservoirs of Ebola virus. Nature. 2005;438:575–57. doi: 10.1038/438575a. [DOI] [PubMed] [Google Scholar]
  11. Lötfering B, Mühlberger E, Tamura T, Klenk H, Becker S. The Nucleoprotein of Marburg Virus Is Target for Multiple Cellular Kinases. Virology. 1999;255:50–62. doi: 10.1006/viro.1998.9577. [DOI] [PubMed] [Google Scholar]
  12. Nakayama E, Yokoyama A, Miyamoto H, Igarashi M, Kishida N, Matsuno K, Marzi A, Feldmann H, Ito K, Saijo M, Takada A. Enzyme-Linked Immunosorbent Assay for Detection of Filovirus Species-Specific Antibodies. Clin Vaccine Immunol. 2010;17:1723–1728. doi: 10.1128/CVI.00170-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Negredo A, Palacios G, Vazquez-Moron S, Gonzalez F, Dopazo H, Molero F, Juste J, Quetglas J, Savji N, de la Cruz M M, Herrera JE, Pizarro M, Hutchison S K, Echevarria J E, Lipkin W I, Tenorio A. Discovery of an ebolavirus-like filovirus in europe. PLoS Pathog. 2011;7:e1002304. doi: 10.1371/journal.ppat.1002304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Niikura M, Ikegami T, Saijo M, Kurane I, Miranda M E, Morikawa S. Detection of Ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein. J Clin Microbiol. 2001;39:3267–3271. doi: 10.1128/JCM.39.9.3267-3271.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Niikura M, Ikegami T, Saijo M, Kurata T, Kurane I, Morikawa S. Analysis of linear B-cell epitopes of the nucleoprotein of ebola virus that distinguish ebola virus subtypes. Clin Diagn Lab Immunol. 2003;10:83–87. doi: 10.1128/CDLI.10.1.83-87.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ou W, Delisle J, Konduru K, Bradfute S, Radoshitzky S R, Retterer C, Kota K, Bavari S, Kuhn J H, Jahrling P B, Kaplan G, Wilsona G A. Development and characterization of rabbit and mouse antibodies against ebolavirus envelope glycoproteins. J Virol Methods. 2011;173:99–109. doi: 10.1016/j.jviromet.2011.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Prehaud C, Hellebrand E, Coudrier D, Volchkov V E, Volchkova VA, Feldmann H, Le Guenno B, Bouloy M. Recombinant Ebola virus nucleoprotein and glycoprotein (Gabon 94 strain) provide new tools for the detection of human infections. J Gen Virol. 1998;79:2565–2572. doi: 10.1099/0022-1317-79-11-2565. [DOI] [PubMed] [Google Scholar]
  18. Saijo M, Niikura M, Morikawa S, Ksiazek T G, Meyer R F, Peters C J, Kurane I. Enzyme-linked immunosorbent assays for detection of antibodies to Ebola and Marburg viruses using recombinant nucleoproteins. J Clin Microbiol. 2001;39:1–7. doi: 10.1128/JCM.39.1.1-7.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Saijo M, Niikura M, Ikegami T, Kurane I, Kurata T, Morikawa S. Laboratory Diagnostic Systems for Ebola and Marburg Hemorrhagic Fevers Developed with Recombinant Proteins. Clin Vaccine Immunol. 2006;13:444–451. doi: 10.1128/CVI.13.4.444-451.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sanchez A, Kiley M P, Klenk H D, Feldmann H. Sequence analysis of the Marburg virus nucleoprotein gene: comparison to Ebola virus and other non-segmented negative-strand RNA viruses. J Gen Virol. 1992;73:347–357. doi: 10.1099/0022-1317-73-2-347. [DOI] [PubMed] [Google Scholar]
  21. Sanchez A, Geisbert T W, Feldmann H. Filoviridae: Marburg and Ebola viruses. In: Knipe D M, Howley P M, editors. Fields Virology. 5th ed. Philadelphia: Lippincott Williams and Wilkins; 2007. pp. 1409–1448. [Google Scholar]
  22. Towner J S, Amman B R, Sealy T K, Carroll S A, Comer J A, Kemp A, Swanepoel R, Paddock C D, Balinandi S, Khristova M L, Formenty P B, Albarino C G, Miller D M, Reed Z D, Kayiwa J T, Mills J N, Cannon D L, Greer P W, Byaruhanga E, Farnon E C, Atimnedi P, Okware S, Katongole-Mbidde E, Downing R, Tappero J W, Zaki S R, Ksiazek T G, Nicho S T, Rollin P E. Isolation of genetically diverse Marburg viruses from Egyptian fruit bats. PLoS Pathog. 2009;5:e1000536. doi: 10.1371/journal.ppat.1000536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. van der Groen G, Kurata T, Mets C. Modifications to indirect immunofluorescence tests on Lassa, Marburg, and Ebola materials. Lancet. 1983;1:654–655. doi: 10.1016/S0140-6736(83)91831-7. [DOI] [PubMed] [Google Scholar]
  24. Watanabe S, Noda T, Kawaoka Y. Functional mapping of the nucleoprotein of Ebola virus. J Virol. 2006;80:3743–3751. doi: 10.1128/JVI.80.8.3743-3751.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. WHO: EBOLA RESPONSE ROADMAP UPDATE 26 September 2014. http://apps.who.int/iris/bitstream/10665/135029/1/roadmapupdate26sept14_eng.pdf.

Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

RESOURCES