Skip to main content
PMC home page
Wiley - PMC COVID-19 Collection logoLink to Wiley - PMC COVID-19 Collection
. 2005 Apr 15;76(2):137–142. doi: 10.1002/jmv.20338

Recombinant protein‐based ELISA and immuno‐cytochemical assay for the diagnosis of SARS†

Alessandra Carattoli 1, Paola Di Bonito 1, Felicia Grasso 1, Colomba Giorgi 1, Francesco Blasi 2, Matthias Niedrig 3, Antonio Cassone 1,✉
PMCID: PMC7166672  PMID: 15834868

Abstract

A new Coronavirus (SARS‐CoV) is the aetiological agent of the severe acute respiratory syndrome (SARS). Because of the critical role played by serological assays for SARS diagnosis, an in‐house ELISA based on SARS‐CoV recombinant antigens was developed. The SARS‐CoV nucleocapsid protein (N), three N fragments (N1, N2, and N3) and the intraviral domain of the membrane protein (M2) were cloned and expressed in Escherichia coli as histidine‐tagged proteins. Six reference sera from SARS patients were used to detect virus‐specific IgG in an ELISA using each recombinant protein as coating antigen. High‐titre positive reactions were detected in all SARS positive sera. The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non‐SARS, low‐tract respiratory infections. Specific hyper‐immune sera to SARS‐CoV and the recombinant proteins, N, N1, N2, N3, and M2 were also generated in mice and rabbits. The specificity of these sera was confirmed by an immunocytochemical assay on biochips of SARS‐CoV infected and uninfected cells. J. Med. Virol. 76:137–142, 2005. © 2005 Wiley‐Liss, Inc.

Keywords: SARS, Coronavirus, diagnosis, antibodies, serology

†

Alessandra Carattoli and Paola Di Bonito equally contributed to the work.

REFERENCES

  1. Chan PK, To WK, Ng KC, Lam RK, Ng TK, Chan RC, Wu A, Yu WC, Lee N, Hui DS, Lai ST, Hon EK, Li CK, Sung JJ, Tam JS. 2004. Laboratory diagnosis of SARS. Emerg Infect Dis 10: 825–831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Che XY, Hao W, Qiu LW, Pan YX, Liao ZY, Xu H, Chen JJ, Hou JL, Woo PC, Lau SK, Kwok YY, Huang Z. 2003. Antibody response of patients with severe acute respiratory syndrome (SARS) to nucleocapsid antigen of SARS‐associated coronavirus. Di Yi Jun Yi Da Xue Xue Bao 23: 637–639. [PubMed] [Google Scholar]
  3. Chen H, Coote B, Attree S, Hiscox JA. 2003. Evaluation of a nucleoprotein‐based enzyme‐linked immunosorbent assay for the detection of antibodies against infectious bronchitis virus. Avian Pathol 32: 519–526. [DOI] [PubMed] [Google Scholar]
  4. Di Bonito P, Nicoletti L, Mochi S, Accardi L, Marchi A, Giorgi C. 1999. Immunological characterization of Toscana virus proteins. Arch Virol 144: 1947–1960. [DOI] [PubMed] [Google Scholar]
  5. Donnelly CA, Fisher MC, Fraser C, Ghani AC, Riley S, Ferguson NM, Anderson RM. 2004. Epidemiological and genetic analysis of severe acute respiratory syndrome. Lancet Infect Dis 4: 672–683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Elia G, Fiermonte G, Pratelli A, Martella V, Camero M, Cirone F, Buonavoglia C. 2003. Recombinant M protein‐based ELISA test for detection of antibodies to canine coronavirus. J Virol Methods 109: 139–142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leung DT, Tam FC, Ma CH, Chan PK, Cheung JL, Niu H, Tam JS, Lim PL. 2004. Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid. J Infect Dis 190: 379–386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liu X, Shi Y, Li P, Li L, Yi Y, Ma Q, Cao C. 2004. Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)‐associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol 11: 227–228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ndifuna A, Waters AK, Zhou M, Collisson EW. 1998. Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus. J Virol Methods 70: 37–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Poon LL, Guan Y, Nicholls JM, Yuen KY, Peiris JS. 2004a. The aetiology, origins, and diagnosis of severe acute respiratory syndrome. Lancet Infect Dis 4: 663–671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Poon LL, Chan KH, Peiris JS. 2004b. Crouching tiger, hidden dragon: The laboratory diagnosis of acute respiratory syndrome. Clin Infect Dis 38: 297–299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Shi Y, Yi Y, Li P, Kuang T, Li L, Dong M, Ma Q, Cao C. 2003. Diagnosis of severe acute respiratory syndrome (SARS) by detection of SARS coronavirus nucleocapsid antibodies in an antigen‐capturing enzyme‐linked immunosorbent assay. J Clin Microbiol 41: 5781–5782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Shi Y, Wan Z, Li L, Li P, Li C, Ma Q, Cao C. 2004a. Antibody responses against SARS‐coronavirus and its nucleocaspid in SARS patients. J Clin Virol 31: 66–68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shi Y, Yi Y, Li P, Kuang T, Li L, Dong M, Ma Q, Cao C. 2004b. Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in SARS patients by enzyme‐linked immunosorbent assay. J Clin Microbiol 42: 2884–2889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, Weissbrich B, Snijder EJ, Rabenau H, Doerr HW, Gorbalenya AE, Ziebuhr J. 2003. Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 84: 2305–2315. [DOI] [PubMed] [Google Scholar]
  16. Woo PC, Lau SK, Wong BH, Tsoi HW, Fung AM, Chan KH, Tam VK, Peiris JS, Yuen KY. 2004. Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 42: 2306–2309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wu HS, Hsieh YC, Su IJ, Lin TH, Chiu SC, Hsu YF, Lin JH, Wang MC, Chen JY, Hsiao PW, Chang GD, Wang AH, Ting HW, Chou CM, Huang CJ. 2004. Early detection of antibodies against various structural proteins of the SARS‐associated coronavirus in SARS patients. J Biomed Sci 11: 117–126. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Medical Virology are provided here courtesy of Wiley

RESOURCES