Characterization of monoclonal antibody against SARS coronavirus nucleocapsid antigen and development of an antigen capture ELISA

Abstract

This report describes the production of several MAbs against N195 protein, a major immunodomain of SARS CoV nucleocapsid protein [He, Q., Chong, K.H., Chang, H.H., Leung, B., Ling, A.E., Wei, T., Chan, S.W., Ooi, E.E., Kwang, J., 2004. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin. Diagn. Lab. Immunol. 11 (2) 417-422.]. One representative IgG1 monoclonal antibody (MAb), S-A5D5, was selected and characterized. S-A5D5 reacted specifically react with both recombinant and native nucleocapsid protein of SARS CoV. The reactivity of S-A5D5 with purified N195 protein and utilization of the MAb as a detector antibody to develop an antigen capture ELISA was assessed. As little as 37.5 pg of purified N protein and 50 TCID(50) of SARS CoV could be detected by the antigen capture ELISA. Specific binding of the MAb S-A5D5 to both purified N195 and SARS CoV nucleocapsid antigen was effectively inhibited by human SARS positive serum and guinea pig anti-N195 serum. The N protein in N195-spike recombinant baculovirus-infected Sf-9 cells could also be identified. N protein was detected in 18 IFA IgM-positive serum samples collected from SARS confirmed patients, but not in nine samples collected from SARS recovery patient. No false positive results were given when 60 samples from healthy individuals were tested, and no cross-reaction occurred when infectious bronchitis virus (IBV), chicken coronavirus, was tested. This monoclonal antibody-based antigen capture ELISA is thus a powerful tool for early diagnosis of SARS CoV infection.

Fig. 1

Fluorescence staining patterns of antibodies in SARS CoV infected Vero cells-based IFA. (A) Monoclonal antibody; (B) polyclonal antibodies; (C) human SARS positive serum; (D–F) side-by-side views of Vero cells staining with MAb, polyclonal antibody and human SARS positive serum under light microscope, respectively.

Fig. 2

IFA fluorescence patterns in N195-spike fusion protein based IFA. (A) Monoclonal antibody; (B) polyclonal antibodies; (C) human SARS positive serum; (D–F) side-by-side views of Sf-9 cells staining with MAb, polyclonal antibody and human SARS positive serum under light microscope, respectively.

Fig. 3

Western blot identification of MAb against N195 protein. A, monoclonal antibody; B, human SARS positive serum; C, non-antibody secreting cell culture (negative control).

Fig. 4

ELISA reactivity of MAb against N195 protein and isolated IgG. (♦) Different dilutions of MAb; (●) supernatant of non-antibody secreting hybridoma; (▴) various concentrations of anti-N195 IgG; (■) IgG from normal guinea pig serum.

Fig. 5

Detection limit of the antigen capture ELISA. (♦) The purified N195; (■) SARS CoV suspension; (▴) recombinant baculovirus infected Sf-9 cells; (×) non-infected Sf-9 cells; (★) non-infected Vero cells.

Fig. 6

Inhibition effects of guinea pig anti-N195 serum and human SARS positive serum on the recognition of nucleocapsid antigen by detector antibody. Groups 1 and 3: 3.75 μg of N195; groups 2 and 4: 0.375 μg of N195; groups 5 and 6: 5 × 10³ and 5 × 10² TCID₅₀ of SARS CoV. The different concentrations of N195 protein or SARS CoV were incubated with guinea pig anti-N195 serum, human SARS positive serum (long bars), guinea pig and human normal serum (short bar), respectively. The contents were subjected to capture ELISA.