Rapid and efficient preparation of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein by immunizing mice.

Che, Xiao-yan; Qiu, Li-wen; Pan, Yu-xian; Xu, Hua; Hao, Wei; Liao, Zhi-yong; Mei, Ya-bo; Zhang, Li-ya; Wan, Zhuo-yue; Yuan, Guo-yong; Huang, Zhen

OBJECTIVE To develop a rapid and efficient method for preparing monoclonal antibodies (mAb) against SARS-associated coronavirus (SARS-Cov) nucleocapsid (N) protein. METHODS BALB/c mice were injected with the recombinant N protein of SARS-Cov into the foot-pads for the immunization, and the popliteal lymph nodes were isolated 15 d later for mAb-producing hybridomas, from which the mAbs against the N protein of SARS-Cov were screened. The identification of the mAb against the N protein of SARS-Cov was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. RESULTS Four strains of hybridomas were obtained that produced the mAb specific to the N protein without detectable cross-reactivity with other pathogens. Of the 4 strains, 2 were identified as the immunoglobulin G1 (IgG1) isotype, 1 IgG2a, and the other IgG2b, with affinity constants (Ka) of 2 of the strains being 4.14 x 10(-9)M and 3.19 x 10(-9)M respectively. CONCLUSION This is the first report on the preparation of mAb that is specific to the SARS-Cov, and the high-specificity and high-affinity mAb produced by the 4 strains of hybridomas provide a basis for further researches on the pathogenesis and early diagnosis of SARS.