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Comparative Study
. 2007 Jul;14(7):839-46.
doi: 10.1128/CVI.00432-06. Epub 2007 May 2.

Comparison of immunoglobulin G responses to the spike and nucleocapsid proteins of severe acute respiratory syndrome (SARS) coronavirus in patients with SARS

Jincun Zhao  1 , Wei Wang, Wenling Wang, Zhendong Zhao, Yan Zhang, Ping Lv, Furong Ren, Xiao-Ming Gao
Affiliations
Comparative Study

Comparison of immunoglobulin G responses to the spike and nucleocapsid proteins of severe acute respiratory syndrome (SARS) coronavirus in patients with SARS

Jincun Zhao et al. Clin Vaccine Immunol. 2007 Jul.

Abstract

Both the nucleocapsid (N) and the spike (S) proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) are able to induce strong humoral responses in humans following an infection. To compare the immunoglobulin G (IgG) responses to the S and N proteins of SARS-CoV in SARS patients during the manifestation/convalescent period with those during the postinfection period, serum samples were collected from hospitalized SARS patients within 6 weeks after the onset of illness (set 1; 57 sequential samples from 19 patients) or 2 to 3 months after their recovery (set 2; 33 postinfection samples from 33 subjects). Serum samples from 100 healthy blood donors (set 3), collected in 2002, were also included. The specific IgG response to whole virus, the fragment from positions 450 to 650 of the S protein (S450-650), and the full-length N protein of SARS-CoV were measured by enzyme-linked immunosorbent assays (ELISAs). Western blot assays were carried out to confirm the ELISA results. Fifty-one of the serum samples in set 1 (89%) bound to the N protein, a proportion similar to that which recognized whole virus (79%) and the S-protein fragment (77%). All 33 serum samples from set 2 were strongly positive for N-protein-specific IgG, while 27 (82%) were positive for anti-S450-650 IgG. Two of the serum samples from set 3 were strongly positive for anti-N-protein IgG but not anti-S450-650 IgG. Similar levels of IgG responses to the S and N proteins were observed in SARS patients during the manifestation and convalescent stages. In the postinfection period, however, a number of patients had much lower serum IgG levels against S450-650 than against the N protein.

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Figures

FIG. 1.
FIG. 1.
SDS-PAGE and Western blot analysis of recombinant proteins. Affinity-purified recombinant N protein (lanes N), S450-650 (lanes S), and 3CL protein (lanes 3CL) were run in two identical SDS-12% polyacrylamide gels. One of the gels was stained with Coomassie blue (A). The protein bands in the unstained gel were transferred onto a nitrocellulose membrane for Western blotting with convalescent-phase serum sample PT18 as the first antibody (B). The detecting antibody was HRP-labeled goat-anti-human IgG. Protein molecular weight markers (lane M) were run in the left-hand lane.
FIG. 2.
FIG. 2.
Sensitivities of the ELISA systems based on N protein and S450-650. ELISA plates were coated with recombinant N protein (A) or S450-650 (B). Convalescent-phase sera from three SARS patients and the pooled HDS control were serially diluted and dispensed, in triplicate, into the wells. HRP-labeled goat anti-human IgG was used as the second antibody, with ortho-phenylenediamine used as the substrate. The results are expressed as the absorbance readings at 492 nm.
FIG. 3.
FIG. 3.
Screening of serum samples from healthy donors. Serum samples from 100 healthy blood donors (set 3) were diluted 1:100 and tested by using the N-protein-based (A) and the S450-650-based (B) ELISAs. The results are expressed as the absorbance readings at 492 nm. The cutoff values were calculated as the mean absorbance readings of the serum samples from all the 100 blood donors plus 3 standard deviations.
FIG. 4.
FIG. 4.
Antibodies against the N protein and S450-650 in patient sera. The N-protein-based (A), S450-650-based (B), and whole-virus-based (C) ELISAs were carried out to analyze the IgG antibodies in serum samples from sets 1 and 2. The results are expressed as the absorbance readings at 492 nm. The same sets of sera were also analyzed by using the Huada ELISA kit, and the results are expressed as the absorbance readings at 450 nm (C). Each bar represents a single serum sample; samples collected at different time points from each individual subject are grouped together. A negative control serum sample (a mixture of serum samples from healthy subjects) was included in each plate for calculation of cutoff values (mean + 3 standard deviations). The cutoff values were 0.30, 0.46, and 0.13 for panels A, B, and C, respectively.
FIG. 5.
FIG. 5.
Correlation among the virus-based, N-protein-based, and S450-650-based ELISA results. The absorbance readings of the virus-based ELISA (OD at 450 nm; serum dilution, 1/11), the N-protein-specific ELISA (OD at 492 nm; serum dilution, 1/100), or the S450-650-specific ELISA (OD at 492 nm; serum dilution, 1/100) were plotted against each other. First-degree regression (r) shows a linear correlation between the results of each two ELISAs. (A) Correlation between virus-based and N-protein-based ELISAs; (B) correlation between virus-based and S450-650-based ELISAs; (C) correlation between N-protein-based and S450-650-based ELISAs.
FIG. 6.
FIG. 6.
Western blot assays of sera from patients and healthy subjects. Recombinant N protein (lanes N), S450-650 (lanes S), and 3CL protein (lanes 3CL) were run in SDS-12% polyacrylamide gels. After electrophoresis, the protein bands were transferred onto nitrocellulose membranes for probing with sera from patients in the convalescent phase (set 2) (A to C) or serum samples N20 and N49 from healthy blood donors (set 3) (D). Bound antibodies were then detected by using HRP-labeled goat anti-human IgG.
FIG. 7.
FIG. 7.
Kinetics of IgG responses in SARS patients. Sequential serum samples from six SARS patients were tested by using the N-protein-based (A), S450-650-based (B), or virus-based (C) ELISA. Sera were diluted 100-fold (A and B) or 11-fold (C), and the results are expressed as the absorbance readings at 492 nm and 450 nm, respectively.
FIG. 8.
FIG. 8.
Neutralization assays. Serially diluted serum samples from patients in the convalescent phase (A) or from healthy blood donors (B) were mixed with medium containing SARS-CoV-S pseudovirus and were incubated for 30 min at 37°C. The mixtures were then distributed into triplicate wells in 96-well plates containing a monolayer of Vero E6 cells seeded at 8 × 103/well the day before. The luciferase activities of the infected cells were determined 48 h later, and the results are expressed as percent infection compared with that for the control group (Vero E6 cells treated only with pseudovirus preparations).
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