动物造模 z-bio 2021-07-06 611

　　Objective: To establish an ELISA detection method for murine coronavirus serum antibodies for daily monitoring of MHV in this area, so as to determine the infection status of SPF mice in time.

　　Method: Select MHV1, MHV-JHM, MHV-A59 3 strains of MHV, purify and concentrate the antigen through L929 cell proliferation and culture, and select the appropriate type of coating antigen. Immunize BALB/C mice to obtain high titer immunopositive serum. Optimize the ELISA reaction system and establish a standardized ELISA kit for the detection of clinical mouse serum samples.

　　Therefore, the MHV coating antigen type suitable for the region was selected as MHV1, and a virus amplification and concentration purification method was established. The prepared MHV antiserum reaches multiple high titer levels in the same batch and can be used as a standardized quality control serum. The best working concentration of coated antigen, test serum and enzyme conjugate is 4.0 μg/mL (10-7.73/0.1 mLTCID50), diluted 1:40 and 1:4000, and the average coefficient of variation within and between batches is. 5.13? 5.57? Detection sensitivity 1:4000 dilution, compatible with mouse Sendai virus (SV), mouse pneumonia virus (PVM), reovir III (Reo3), murine parvovirus (MVM), murine pox. Virus (Ect) positive serum does not cross-react. The relative deviation of the stability test was less than 10? Test 165 serum samples, the coincidence rate of positive serum was 97.37?37/38), and the coincidence rate of negative serum was 92.19?118/127).

　　Conclusion: The ELISA method established in this study has high specificity, sensitivity and reproducibility for detecting MHV antibodies in mouse serum. It can be used for daily pathogenic monitoring of MHV in this field and can accurately determine the infection status of mice.