Preparation And Preliminary Application Of Monoclonal Antibodies Against The Spike And Nucleocapsid Protein Of SARS Associated Coronavirus

Severe Acute Respiratory Syndrome (SARS), the first severe infectious disease to emerge in the twenty-first century, has spread at a rapid pace around the Globe. World Health Organization announced that SARS is due to an infection with a novel coronavirus which was termed SARS associated coronavirus (SARS-CoV). SARS has also adversely effected economic growth, trade, tourism, business and industrial performance, and social stability as well as public health. It is considered important to develop rapid, valid and consistent diagnostic tests for detecting the virus in early stages of infection.This study aimed to prepare monoclonal antibodies (mAbs) against recombinant spike(S) and nucleocapsid (N) protein of SARS-CoV as well as in order to develop sandwich ELISA measures for detecting the SARS-CoV.In this study, the recombinant S protein and N protein of SARS-CoV expressed in E. coli were used to immunize BALB/c mice. The mAbs against S or N protein were prepared by routine hybridoma technology. Several mAb characteristics were identified, including chromosome analysis, stability of secreting, immunoglobulin (Ig) isotype, titers, specificity, neutralization capacity and antigen-binding epitope of mAb against S protein. Antibodies against S or N protein were used to developsandwich ELISA measure to detect SARS-CoV using serum samples from healthy donors (180 samples) and clinically diagnosed SARS patients (24 samples, 12 positive and 12 negative IgG antibodies respectively).Three hybridoma cell lines secreting mAbs against S protein (B8G2, B10F6, C10G10) and two hybridoma cell lines secreting mAbs against N protein (G5H12G12, F2G6G3) were established. The mean number of hybridoma chromosomes was near the sum of the two parent cells'. The titers of mAbs in supernatant and ascites were both high. The mAb-G5H12G12 Ig class was IgG2a (k), and the others were IgGl (k). The specificity was strong and the neutralization titer of the anti-S protein mAb was 1:25. With sandwich ELISA assay, no positive results were detected in any serum samples of healthy donors and the samples from clinically diagnosed SARS patients who had SARS specific IgG antibodies. Among the serum samples from clinically diagnosed SARS patients but IgG antibodies negative, there were 4 positive results obtained by using sandwich ELISA with antibodies against N protein, and all the positive cases were within 10 days after the onset of the clinical presentation. But no positive results were detected by using sandwich ELISA with anti-S recombination protein antibodies.In conclusion, we have successfully prepared mAbs respectively against recombinant spike and nucleocapsid proteins of SARS-CoV, and developed sandwich ELISA measures with antibodies against N protein which can be used for early clinical diagnosis.