| Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneunoniae,a disease characterized by high morbidity and low mortality,and causes a severe economical loss worldwide.Mhp strain 168 was selected to prepare a monoclonal antibody to develop a indirect competitive enzyme-linked immunosorbant assay(Ci-ELISA) which could satisfy the clinical demand in this study.Mhp strain 168 F323 grown in KM2 medium was harvested by centrifugation and suspended in PBS. The whole cell protein was prepared by freeze thawing and ultrasonic treatment.the BALB/c mice were immuned with purified whole cell protein.the mycloma cell line Sp2/0 was fused with the spleen of immuned BALB/c mice.the specific monoclonal antibody were screened by indirect ELISA and IHA,and one hybridoma cell lines steadily secreting monoclonal antibody(McAb) against Mhp was obtained by three limiting dilution,and was named 3G5.the titer of the antibody in the cell supernatant and ascites were 1:128 and 1:6400 respectively.the Ig of the monoclonal antibody belonged to IgG-subgroup.the results of western-blot test indicated that the antibody could react with the 70kD protein of Mhp.The ascites was purified by ammonium sulfate precipitation,protein concentration was 4.37mg/mL.In order to detect the antibodies against Mhp,a Ci-ELISA based on whole cell antigen and monoclonal antibody was developed.the optimiun condition of competitive ELISA were developed as follows:15μg/μL whole cell protein of Mhp in carbonate sodium buffer was used to coat ELISA plates;1:400 dilution of working titer for McAb;1:5000 dilution of working titer for goat anti-mouse IgG.the results showed that the competitive ELISA had high specificity,sensitivity and repetition.28 clinical pig sera were detected by Ci-ELISA and test kit for the antibody against Mhp,the results showed that the agreement ratio between the two methods was 82.1%.It indicated that McAb against Mhp should be helpful in rapid detection of Mhp. |
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