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. 2005 May;33(1):12-8.
doi: 10.1016/j.jcv.2004.09.024.

Development and evaluation of an enzyme-linked immunosorbent assay for detection of antibodies against the spike protein of SARS-coronavirus

Jincun Zhao  1 , Wei Wang, Guang-Fa Wang, Yonghua Li, Hui Zhuang, Xiaoyuan Xu, Furong Ren, Zhendong Zhao, Xiao-Ming Gao
Affiliations

Development and evaluation of an enzyme-linked immunosorbent assay for detection of antibodies against the spike protein of SARS-coronavirus

Jincun Zhao et al. J Clin Virol. 2005 May.

Abstract

Background: Severe acute respiratory syndrome (SARS) is caused by infection with SARS-associated coronavirus (CoV). Amino acid residues 450-650 of the spike (S) glycoprotein of SARS-CoV (S450-650) contains dominant epitopes for anti-viral antibodies (Abs) in patient sera.

Objectives: To develop and evaluate an ELISA system for detection of anti-S Abs in patient sera.

Study design: Express recombinant S450-650 in E. Coli and evaluate the sensitivity and specificity of an ELISA system based on the S450-650 polypeptide.

Results: The S450-650-based ELISA detected IgG Abs in 41 out of 51 serum samples from 22 hospitalized patients with probable SARS, a result closely correlated with that obtained with a virus-based ELISA (r = 0.75, k = 0.8). Differential anti-S IgG responses were observed amongst SARS patients. Some of them produced anti-S Abs early during their infection, while others failed to make IgG Abs against the S450-650 polypeptide. None of the serum samples from 100 healthy blood donors was positive in the S450-650-based assay.

Conclusion: The S450-650-based ELISA can detect anti-S IgG Abs with high sensitivity and specificity.

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Figures

Fig. 1
Fig. 1
SDS-PAGE and WB analysis of the recombinant S450-650 fragment. Affinity purified recombinant S450-650 was run in 2 identical SDS PAGE 12% gels with molecular weight markers in Lane M. One of the gels was stained with Coomassie blue (Lanes M and A). Protein bands in the other gel were transferred onto cellulose nitrite membranes for WB with convalescent serum from patient PT-LX (Lane B), or anti-His-tag mAb (Lane C), or serum from a healthy subject (Lane D), as the first Ab.
Fig. 2
Fig. 2
Sensitivity of the S450-650-based ELISA. ELISA plates were coated with recombinant S450-650. Serum samples from 3 convalescent SARS patients (▴, ♢, ▵) and 2 healthy individuals (■, □) were serial diluted and dispensed, in triplicates, into the wells. HRP-labeled goat anti-human IgG was used as secondary Ab with OPD as substrate. The results are expressed as absorbance reading at 492 nm wavelength.
Fig. 3
Fig. 3
Comparison of S450-650-based and virus-based ELISA results. The S450-650-based ELISA kit was used to screen serum samples from (A) 100 healthy blood donors and (B) 18 patients (PT01 to PT18, collected between 10 and 42 days from the onset of illness) with probable SARS. The serum samples were 100 folds diluted, results are expressed as absorbance reading at 492 nm wavelength. Cutoff value was 0.46. ELISA results obtained with the Huada kit are shown in (C) for comparison. In this case, serum samples were 10 folds diluted, results are expressed as absorbance reading at wavelength of 450 nm. Cutoff value was 0.139. Each bar represents one serum sample, samples from each patient were group together in the order of collection time (days) after onset of illness.
Fig. 4
Fig. 4
Correlation between S450-650-based and SARS-CoV-based ELISA results. The ELISA results shown in Fig. 3B and 3C were plotted against each other. First-degree regression (r = 0.75) shows a linear correlation between the results of the two ELISA tests.
Fig. 5
Fig. 5
Time course of Ab responses against S450-650 and SARS-CoV in patients. Sequential serum samples from seven patients were tested using the (A and C) S450-650-based and (B and D) Huada ELISA kits. Serum samples were diluted 100 folds in (A) and (C), 10 folds in (B) and (D), and the results are expressed as absorbance reading at 492 or 450 nm, respectively. Cutoff values were (A and C) 0.46 or (B and D) 0.18.
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