Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
J Med Virol. 2004 Jul; 73(3): 338–346.
Published online 2004 May 24. doi: 10.1002/jmv.20096
PMCID: PMC7167198
PMID: 15170626

Evaluation of antibody responses against SARS coronaviral nucleocapsid or spike proteins by immunoblotting or ELISA

Li‐Rung Huang, 1 Chi‐Ming Chiu, 2 Shiou‐Hwei Yeh, 3 Wen‐Hung Huang, 2 Po‐Ren Hsueh, 4 Wen‐Zieh Yang, 5 Jyh‐Yuan Yang, 5 Ih‐Jen Su, 5 Shan‐Chwen Chang, 4 and Pei‐Jer Chencorresponding author 1 , 2 , 4
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

Severe acute respiratory syndrome (SARS)‐CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. To study the humoral responses against SARS‐CoV, we evaluated nucleocapsid (N) and spike (S) proteins‐specific antibodies in patients' sera by Western blotting and enzyme‐linked immunosorbent assay (ELISA). Recombinant N and S proteins of SARS‐CoV were purified from transformed E. coli. Serum specimens from 40 SARS‐CoV‐infected patients in the convalescent phase were analyzed by Western blotting using the purified antigens. Serial serum specimens from 12 RT‐PCR‐confirmed SARS patients were assayed by ELISA using the recombinant N protein as coated antigen. By Western blotting, 97.5% of the SARS patients were positive for N protein‐specific antibodies whereas only 47.5% of the samples were positive for S protein‐specific antibodies. Using N protein‐based ELISA, 10 out of the 12 patients were positive for N protein‐specific antibodies and 6 of them showed seroconversion at mean of 16 days after onset of fever. Immunoblotting was useful for detecting the humoral immune response after SARS‐CoV infection. Antibodies against SARS‐CoV N protein appear at the early stage of infection, therefore, N protein‐based ELISA could serve as a simple, sensitive, and specific test for diagnosing SARS‐CoV infection. J. Med. Virol. 73:338–346, 2004. © 2004 Wiley‐Liss, Inc.

Keywords: SARS, SARS‐CoV, ELISA, N protein, S protein

REFERENCES

  • Baric RS, Nelson GW, Fleming JO, Deans RJ, Keck JG, Casteel N, Stohlman SA. 1988. Interactions between coronavirus nucleocapsid protein and viral RNAs: Implications for viral transcription. J Virol 62: 4280–4287. [PMC free article] [PubMed] [Google Scholar]
  • Bergmann CC, Yao Q, Lin M, Stohlman SA. 1996. The JHM strain of mouse hepatitis virus induces a spike protein‐specific Db‐restricted cytotoxic T cell response. J Gen Virol 77: 315–325. [PubMed] [Google Scholar]
  • Boots AM, Van Lierop MJ, Kusters JG, Van Kooten PJ, Van der Zeijst BA, Hensen EJ. 1991. MHC class II‐restricted T‐cell hybridomas recognizing the nucleocapsid protein of avian coronavirus IBV. Immunology 72: 10–14. [PMC free article] [PubMed] [Google Scholar]
  • Boots AM, Benaissa‐Trouw BJ, Hesselink W, Rijke E, Schrier C, Hensen EJ. 1992. Induction of anti‐viral immune responses by immunization with recombinant‐DNA encoded avian coronavirus nucleocapsid protein. Vaccine 10: 119–124. [PMC free article] [PubMed] [Google Scholar]
  • Bradburne AF, Somerset BA. 1972. Coronative antibody tires in sera of healthy adults and experimentally infected volunteers. J Hyg (Lond) 70: 235–244. [PMC free article] [PubMed] [Google Scholar]
  • Collins AR, Knobler RL, Powell H, Buchmeier MJ. 1982. Monoclonal antibodies to murine hepatitis virus‐4 (strain JHM) define the viral glycoprotein responsible for attachment and cell–cell fusion. Virology 119: 358–371. [PMC free article] [PubMed] [Google Scholar]
  • Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348: 1967–1976. [PubMed] [Google Scholar]
  • Fleming JO, Stohlman SA, Harmon RC, Lai MM, Frelinger JA, Weiner LP. 1983. Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV‐4) virus. Virology 131: 296–307. [PMC free article] [PubMed] [Google Scholar]
  • Gerber JD, Ingersoll JD, Gast AM, Christianson KK, Selzer NL, Landon RM, Pfeiffer NE, Sharpee RL, Beckenhauer WH. 1990. Protection against feline infectious peritonitis by intranasal inoculation of a temperature‐sensitive FIPV vaccine. Vaccine 8: 536–542. [PMC free article] [PubMed] [Google Scholar]
  • Hsueh PR, Hsiao CH, Yeh SH, Wang WK, Chen PJ, Wang JT, Chang SC, Kao CL, Yang PC. 2003. Microbiologic characteristics, serologic responses, and clinical manifestations in severe acute respiratory syndrome, Taiwan. Emerg Infect Dis 9: 1163–1167. [PMC free article] [PubMed] [Google Scholar]
  • Krokhin O, Li Y, Andonov A, Feldmann H, Flick R, Jones S, Stroeher U, Bastien N, Dasuri KV, Cheng K, Simonsen JN, Perreault H, Wilkins J, Ens W, Plummer F, Standing KG. 2003. Mass spectrometric characterization of proteins from the SARS virus: A preliminary report. Mol Cell Proteomics 2: 346–356. [PMC free article] [PubMed] [Google Scholar]
  • Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348: 1953–1966. [PubMed] [Google Scholar]
  • Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, van Amerongen G, van Riel D, Laman JD, de Jong T, van Doornum G, Lim W, Ling AE, Chan PK, Tam JS, Zambon MC, Gopal R, Drosten C, van der Werf S, Escriou N, Manuguerra JC, Stohr K, Peiris JS, Osterhaus AD. 2003. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362: 263–270. [PMC free article] [PubMed] [Google Scholar]
  • Lai MMC, Holmes KV. 2001. Coronaviridae: The viruses and their replication In: Knipe D, editor. Fields Virology. Philadelphia, Pennsylvania, USA: Lippincott Williams &Wilkins; pp 1163–1185. [Google Scholar]
  • Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran M, Sullivan JL, Luzuriaga K, Greenough TC, Choe H, Farzan M. 2003. Angiotensin‐converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426: 450–454. [PMC free article] [PubMed] [Google Scholar]
  • Liu C, Kokuho T, Kubota T, Watanabe S, Inumaru S, Yokomizo Y, Onodera T. 2001. DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus. Virus Res 80: 75–82. [PubMed] [Google Scholar]
  • Macnaughton MR, Hasony HJ, Madge MH, Reed SE. 1981. Antibody to virus components in volunteers experimentally infected with human coronavirus 229E group viruses. Infect Immun 31: 845–849. [PMC free article] [PubMed] [Google Scholar]
  • McIntosh K, Kapikian AZ, Turner HC, Hartley JW, Parrott RH, Ahanock RM. 1970. Seroepidemiologic studies of coronavirus infection in adults and children. Am J Epidemiol 91: 585–592. [PMC free article] [PubMed] [Google Scholar]
  • Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, Law KI, Tang BS, Hon TY, Chan CS, Chan KH, Ng JS, Zheng BJ, Ng WL, Lai RW, Guan Y, Yuen KY. 2003a. Clinical progression and viral load in a community outbreak of coronavirus‐associated SARS pneumonia: A prospective study. Lancet 361: 1767–1772. [PMC free article] [PubMed] [Google Scholar]
  • Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, Nicholls J, Yee WK, Yan WW, Cheung MT, Cheng VC, Chan KH, Tsang DN, Yung RW, Ng TK, Yuen KY. 2003b. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361: 1319–1325. [PMC free article] [PubMed] [Google Scholar]
  • Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, Penaranda S, Bankamp B, Maher K, Chen MH, Tong S, Tamin A, Lowe L, Frace M, DeRisi JL, Chen Q, Wang D, Erdman DD, Peret TC, Burns C, Ksiazek TG, Rollin PE, Sanchez A, Liffick S, Holloway B, Limor J, McCaustland K, Olsen‐Rasmussen M, Fouchier R, Gunther S, Osterhaus AD, Drosten C, Pallansch MA, Anderson LJ, Bellini WJ. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300: 1394–1399. [PubMed] [Google Scholar]
  • Sambrook J, Russell DW. 2001. Commonly used techniques in molecular cloning. Molecular cloning, 3rd edn. New York: Cold Spring Harbor Laboratory Press; pp A8.40–A48.54. [Google Scholar]
  • Stohlman SA, Kyuwa S, Cohen M, Bergmann C, Polo JM, Yeh J, Anthony R, Keck JG. 1992. Mouse hepatitis virus nucleocapsid protein‐specific cytotoxic T lymphocytes are Ld restricted and specific for the carboxy terminus. Virology 189: 217–224. [PMC free article] [PubMed] [Google Scholar]
  • Tuboly T, Nagy E, Derbyshire JB. 1995. Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies. Can J Vet Res 59: 70–72. [PMC free article] [PubMed] [Google Scholar]
  • Wege H, Schliephake A, Korner H, Flory E. 1993. An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis. J Gen Virol 74: 1287–1294. [PubMed] [Google Scholar]
  • WHO . 2003. Cumulative number of reported probable cases of sever acute respiratory syndrome. http://www.whoint/csr/sars/country/2003_07_09/en/ accessed July 10, 2003.
  • Yeh SH, Wang HY, Tsai CY, Kao CL, Yang JY, Liu HW, Su IJ, Tsai SF, Chen DS, Chen PJ, Lee YT, Teng CM, Yang PC, Ho HN, Chang MF, Wang JT, Chang SC, Wang WK, Hsiao CH, Hsueh PR. 2004. Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: Molecular epidemiology and genome evolution. Proc Natl Acad Sci USA 101: 2542–2547. [PMC free article] [PubMed] [Google Scholar]
Articles from Journal of Medical Virology are provided here courtesy of Wiley
-