Identification And Application Of The Monoclonal Antibodies Against SARS-CoV

The severe acute respiratory syndrome (SARS) is a highly virulent emerging disease. The etiological agent of SARS was identified as a new coronavirus(SARS-CoV). Although the first SARS epidemic was successfully controlled, it remains a potential threat to human due to its mysterious source of initial infection, its unknown animal host and unknown route of transmission. As the symptoms of SARS can be confused clinically with many respiratory diseases caused by other etiological agent, the early laboratory diagnosis became more important. Furthermore, the current medical strategies for SARS mainly relying on non-specific anti-viral and supportive treatment, and the passive immune therapy by using serum of convalescent patients has been proved clinically effective, therefore more attention has been paid to develop the passive treatment reagents.In order to develop early and specific laboratory diagnostic test and the passive immunity reagents, we immunized BALB/C mice with purified and inactivated SARS-CoV (BJ-01) and established 12 hybridoma cell lines which could stably secret anti-SARS monoclonal antibodies (mAbs). Conventional immunology identification showed that: 1. Detection of antibody titres by IFA found that apart from 1D4, titres of the other 11 mAbs were between 1: 320-1: 20480. This results indicated that these mAbs all had high anti-SARS activities and they could react with viral strains isolated from different regions; 2: Specific identification of type and subtype of immunoglobulin by double agar diffusion and ELISA demonstrated that the heavy chain of 5 mAbs (2D4,1A2,2C5,2A3,3B4) were belong to IgG1, and 5 (1B4,2B1,1C5,1A5,2A2) were belong to IgG2a, 1 (3A3) was IgG2b and another 1 (1D4) was IgM. All the light chains belong to k; 3: The neutralizing activity test of the 12 mAbs with micro-culture showed that 6 of them (1A2,1A5,2D4,2A3,2C5,3B4) hadneutralizing activities, wherein the neutralizing activities of 1A5 and 2C5 were 1:20480 and 1:5957 respectively.Based on the above identifications, we further analyzed the antigen-binding epitopes of these mAbs. First, recombinant expressed S and N proteins were employed to screen the mAbs . The results displayed that 6 mAbs could recognize S protein (1A5,2C5,2A3,1B4,2B1,3A3) and 2 could recognize N protein (2D4,1A2). This indicated that their antigen-binding epitopes were located on S and N proteins respectively. Another 4 mAbs couldn't recognize either S or N proteins without any pre-treatment. However, 1 of them (2D4) could react with DTT-treated SI fragment by Western-blot assay. This further illustrated that the antigen epitope of 2D4 could be exposed and to react with 2D4 only after the denaturizing treatment. Another 3 mAbs might recognize proteins other than S and N. The antigen specificity of 7 mAbs specific to S protein was further mapped by using S fragments with different length. 4 mAbs (2A3,1B4,2B1,2C5) showed high reactivity against SI domain (aa 12-311) and 2 mAbs were against aa310-535 of SI domain. The latter was reported to be the receptor-binding d omain ( RBD) o f S ARS-CoV. T he e pitope o f 3 A3 w as m apped t o aa797-l 192 of S2 domain.Among the 6 mAbs with neutralizing activities, 3 (1A5,2A3,2C5) were specific to S protein and 2 (2D4,1A2) were specific to N protein, another 1(3B4) was against other structural protein. These results showed that both S and N protein had neutralizing antigen epitopes. The neutralizing activities specific to S protein were higher than to N protein. The epitope mapping of 2 mAbs (lA5,2C5)with high neutralizing activities found that both of their antigen binding sites were located in RBD of SARS-CoV. This further indicated that the antigen epitopes of this domain were important protective epitopes.By using these mAbs, VeroE6 cells infected with SARS-CoV were analyzed with IFA and lung> bronchus and lymphaden specimen from Macaca rhesus inoculated with SARS-CoV were analyzed with immunohistochemistry, virus in these specimen could all be detected by the mAbs. These results confirmed the specificity and affinity of these mAbs.