Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus

Abstract

Severe acute respiratory syndrome-coronavirus (SARS-CoV) causes an infectious disease through respiratory route. Diagnosing the disease effectively and accurately at early stage is essential for preventing the disease transmission and performing antiviral treatment. In this study, we raised monoclonal antibodies (mAbs) against the nucleocapsid (N) protein of SARS-CoV and mapped epitopes by using different truncated N protein fragments. The mapping of those epitopes was valuable for constructing pair-Abs used in serological diagnosis. The results showed that all of the six raised mAbs were divided into two groups recognizing the region of amino acids 249-317 (A group) or 317-395 (B group). This region spanning amino acids 249-395 contains predominant B cell epitopes located at the C-terminus of N protein. One pair-Abs, consisting of N protein-specific rabbit polyclonal antibody and SARS-CoV N protein-specific mAb, was selected to construct a sandwich ELISA-kit. The kit was able to specifically detect SARS-CoV N proteins in serum samples.

Fig. 1

Structure of truncated N protein fragments. The numbers at both ends of the line indicate the starting and ending amino acid residue position in the fragments.

Fig. 2

Characterization of mAbs by ELISA with full-length N protein and truncated N protein fragments (N1–N4). N protein and N1–N4 fragments were coated on a 96-well ELISA plate. After blocking with 10% bovine serum, the plate was incubated with different monoclonal antibodies. Then bound antibodies were detected with HRP-conjugated goat anti-mouse IgG antibody and the OD values were measured at 450 nm. Data points represent means (error bars, standard deviations). (A) Reaction with two mAbs (A group). (B) Reaction with four mAbs (B group).

Fig. 3

Affinity of the selected mAbs. Affinity constant was measured as described in Materials and methods. (A) Affinity for S-39-2 mAb (3.1 × 10⁹ L/mol). (B) Affinity for N-17-3 mAb (7.2 × 10⁸ L/mol).

Fig. 4

Western blotting to detect N protein. (A) with N-17-3 monoclonal antibody; (B) with S-39-2 monoclonal antibody. The E. coli-expressed N protein, the lysates of SARS-CoV-infected Vero E6 cells and uninfected Vero E6 cells (control) were subjected to SDS–PAGE and then transferred to PVDF membrane. The blots were probed with different monoclonal antibodies and detected with HRP-conjugated goat anti-mouse IgG antibody.

Fig. 5

Immunoreactivity of anti-N protein antibodies with four human coronaviruses. Reactivity to other three coronavirus N proteins (HCoV-OC43, 229E, and NL63) was detected by SARS-CoV N protein-specific Abs by Western blot. (A) Detected with N-17-3 mAb (1:1000). (B) Detected with S-39-2 mAb (1:1000) and (C) detected with N protein pAb (1:80,000). In each panel, five samples were tested. Control means normal Vero E6 cell lysates. SARS means SARS-CoV-infected Vero E6 cell lysates. Other three are from OC43, 229E, and NL63-infected host cell lysates.

Fig. 6

Reactivity of pair-Abs with N protein. N-17-3 mAb or S-39-2 mAb or the mixture of N-17-3 mAb and S-39-2 mAb was selected as a coating Ab on the 96-well plate, and then different concentrations of the N protein were added into the plate. HRP-pAb (1:500) was used to detect the N protein.

Fig. 7

Detection curve for N protein and JC protein by sandwich ELISA. Monoclonal antibody S-39-2 (5 μg/ml) was coated on an ELISA plate. Serial dilutions of N protein and JC protein were detected by HRP-pAb (1:500).