Preparation And Identification Of Monoclonal Antibody Against Mycoplasma Hyopneumoniae

Mycoplasma hyopneumoniae (Mhp) is the causative agent of Swine Enzootic Pneumoniae (SEP), a disease found on pig farms worldwide which is characterized by high morbidity and low mortality rates. Since Mhp can compromise the mucociliary clearance mechanism of respiratory, thus predispose the pigs to secondary pulmonary infections and increase the mortality. Now, the early detection of Mhp infection is difficult and there is also no effective drugs to control prevail of this disease. In this experiment, Mhp strain 168 was selected to prepare monoclonal antibodies, which should be potentially used for Mhp infection. And screen a method to detect Mhp by enzyme-linked immunosorbent assay.Mhp strain I68F323, grown in KM2 medium which contain horse serum, were harvested by super-centrifugation and suspended in PBS. The 8-week-old female BALB/c mice were injected intraperitoneally with 200ug whole membrane proteins of freeze-thaw and ultrasonic processor broken killed Mhp, emulsified in Freund's complete adjuvant for the first and in Freund's incomplete adjuvant for the three following injections. Injections were given at 2-week intervals, followed by an intravenous dose of the antigen without adjuvant 3 days prior to the fusion experiment. After the spleen cells from immunized mice were fused with Sp2/0 myeloma cells, the hybridoma cells were screened by indirect ELISA and the positive clones were subcloned three times by serial dilutions. Two hybridoma cell lines secreting monoclonal antibodies (McAb) designed as 2G11,4E7 were established. The subtypes of the McAb were all IgG3 and titers of their ascetic fluids were all about 1:10000 in indirect ELISA. The immunoblotting result indicated that the McAb could react with 70KD and 40KD protein of Mhp, respectively, and couldn't bind with control serum samples. The results suggested that the McAb were all specific to the protein of Mhp.An indirect enzyme-linked immunosorbent assay for detecting serum antibodies" to the Mycoplasma hyopneumoniae (Mhp) was established by using Mhp strain 168 as antigen for coating.The indirect ELISA should be performed as following: The working concentration of the antigen coated by PBS (pH 9.6) should be 1.5ug/ml, and the optimum diluted concentration of the tested serum should be 1:100. After the action for 90 minutes, suspension should be performed by 2% gelatin. PBS containing 0.5%Tween-20 should be used as wash solution and diluting agent. And the optimum action time of the enzyme-signed antibodies, which were diluted by 1:10000, should be 60 minutes.Contrasting with the result of the monoclonal blocking ELISA detection, find out monoclonal blocking ELISA detect forepart infect have good specificity, sensitivity.