关键词: 小鼠肝炎病毒, 血清学, 酶联免疫吸附实验（ELISA）

Abstract: Objective To establish the Mouse Hepatitis Virus (MHV) serum antibody ELISA test method for routine monitoring of MHV locally, in order to make timely judgments on the infection status of SPF mice. Methods Three kinds of MHV strains (MHV1, MHV-JHM and MHV-A594) were amplified in the L929 cells and purified by ultracentrifugation method. The appropriate virus antigen type was determined and an enzyme-linked immunosorbent assay (ELISA) system was established. SPF BALB/c mice were immunized to obtain high titer immunopositive serum. The purified MHV antigens were coated in microtiter plates and the detection system of ELISA flat was also optimized to apply clinical mice serum detection．Results The suitable coating antigen for local region was MHV1 and the methods for virus amplification and antigen purification were established. The prepared MHV antiserum keep high titer and can be used as standardized quality control serum. The optimal working concentrations of coated antigen, serum to be tested and enzyme conjugate were 4.0μg/mL (10-7.73/0.1mL TCID50), 1: 40 and 1:4000 respectively. The inter-assay and intra-assay average coefficient of variation is 5.13% and 5.57%, respectively. The detection sensitivity reach more than 1: 4000. There was no cross-reactivity with Sendai Virus (SV), Pneumonia Virus of Mice (PVM), Reovirus type III (Reo3), Minute Virus of Mice (MVM) and Ectromelia Virus (Ect). The relative deviation of stability test was less than 10%. A totally 165 sera samples were analyzed by the current ELISA method, the positive serum coincidence rate was 97.37% (37/38) and the negative serum coincidence rate was 92.19% (118/127), compared with the result of the commercial ELISA kit. Conclusion We have established ELISA method detection of mouse serum MHV, with high specificity, sensitivity and reproducibility It can be used for daily pathogen monitoring of MHV in this region, so as to make accurate judgment on the infection status of mice.