PREPARATION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SARS-ASSOCIATED CORONAVIRUS NUCLEOCAPSID PROTEIN

Objective To prepare monoclonal antibodies against recombinant severe acute respiratory syndrome(SARS)-associated coronavirus(CoV) nucleocapsid(N) protein in order to offer a tool for either further research or early diagnosis of SARS. Method BALB/c mice were immunized by injection of recombinant N protein of SARS-CoV. The hybridomas which can produce McAbs were plated in a semi-solid medium containing methycellulose. The identifications of the mAb against the N protein of SARS-CoV, including spcificity, affinity, isotypes and results of pairing test, were performed using indirect enzyme-linked immunosorbent assay(ELISA), indirect fluorescent antibody assay(IFA), and double diffusion assay. Results Fifteen strains of hybridomas were obtained, 10 of them were specific to SARS-CoV. Affinity constants(Ka) was between 10 8mol/L -1and 10 9mol/L -1. Among the 10 strains, 1 strain was identified as the immunoglobulin G 2b(IgG 2b) isotype, another one was IgG 3,and the others were IgG 1. Eight out of the 10 strains were specific to the N protein without detectable cross-reactivity with twelve related pathogens. Two strains had cross reactions. One reacted with influenza A,B, and parainfluenza serotypes 1,2,3, another one reacted with influenza A and B. Five out of 10 formed six pair ways, the sensityvity of two pairs reached 1μg/L. ConclusionWe obtained McAbs of high-specificity and high-affinity to SARS-CoV and established the double-antibody sandwich ELISA-based test for detecting N antigen. The results of our work have provided a foundation for further research on either protein group or early diagnosis of SARS.