Preparation of monoclonal antibodies against SARS coronavirus and staining usage in lung autopsy specimens

Abstract

To prepare monoclonal antibodies against SARS coronavirus (SARS-CoV) on the purpose to explore the diagnosis methods of SARS. Female BALB/C mice were immunized with disinfected SARS-CoV (PUMC01) and the spleen cells were fused with myeloma NS-1 cells. The hybridoma cell strains were screened by enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA) and Western blotting. Autopsy lung tissue sections from SARS patients were stained with ascites of monoclonal antibody (M2 strain) by immunohistochemical technique. Six strains of hybridomas that produced the monoclonal antibodies against SARS-CoV were obtained. The hybridomas were tested to have specific reactions with SARS-CoV and no cross-reactivates with other common respiratory disease causing pathogens. Of the 6 strains, 1 was identified as the immunoglobulin G3 (IgG3) isotype, 5 were IgG1. Western blotting showed that one strain (M2) reacted with 68000- Dalton (68KD) protein and four strains with 27000-dalton (27KD) protein. Band of M4 stain was not got by western blotting. Brown particles were seen in macrophages and pneumocytes in autopsy lung tissues from SARS patients. Monoclonal antibodies we made were specific to the SARS-CoV, and they had been used to detect SARS-CoV in autopsy lung tissues specimens with positive results.