Production Of Monoclonal Antibodies And Establishment Of Blocking ELISA For Detection Of Chlamydophila Abortus In Swine

Chlamydophila abortus (C.abortus) is an important zoonotic pathogen with broad host spectrums, which mainly causes the reproductive disorders, particularly abortion and endometritis. Recent years, prevalence of C. abortus in swine had been reported in lots of countries. Efficient vaccine had not been used, so antibody detection is still an effective method to monitor and diagnose the infection. In this study, a riped and simple Blocking ELISA for detection of antibodies against C.abortu was developed.1. Cloning and expression of MOMP protein of C. abortusComplete momp gene of C.abortus HB1043strain was amplified by PCR and cloned into pMD18-T vector. The gene sequence was analyzed and the immunologic fragment of271-1176bp was cloned into pET-28a vector. The recombinant pET28a-MOMP plasmid was transformated in BL21to express. The protein was purified with Ni sepharose6fast flow and its reactivity was detected by Western-blot.2. Production of monoclonal antibodies(McAbs) against MOMP6-week BALB/c mice were immunized with the immunogen which prepared by mixing the formaldehyde-inactived C. abortus-infected chicken embryo yolk sac supernatant fluid with the Freund’s adjuvant. The indirect ELISA based on rMOMP protein was used to monitor the antibody titer,35days after immunized, the mice which had the tallest antibody titer was selected to fuse. Hybridoma supernatants for antibody production were screened by indirect rMOMP-ELISA. After3times cloning,5hybridoma cell lines secreting monoclonal antibodies(McAbs) aginst MOMP were selected and named as5D7,1D3,4E5,1E6, and4C9. The further studies indicated that the subtypes of5D7and1E6were IgGl and IgG3, respectively; the subtypes of others were IgG2b. The antibody titers of the ascites fluids were105-106. The McAbs were purified by octylic acid ammonium sulfate method and were labeled with HRP.3. Establishment of the Blocking ELISAThe Blocking ELISA was established by using the rMOMP as the coating antigen and using the HPR-labeled McAb4C9as the competive antibody. The most suitable diluted concentration of the coating antigen, serum sample and the HPR-labeled McAb were1:80,1:2and1:32000, respectively. The CUT-OFF value was0.377.261serum samples were detected with the IHA and the Blocking ELISA, and the diagnosis coincidence rate of the two methods was78.5%.