Establish Of Double Antigens Sandwich ELISA Based On Chlamydia Abortus MIP And Preparation Of Monoclonal Antibodies

Chlamydia abortus(C. abortus) is a kind of obligate intracellular Gram-negative bacteria which can infect female animals and lead toabortion, stillborn; it can also infect male animals and cause clinical snydrome such as orchitis, urethritis; pregnant women contact with infected animals directly can also been infected and abortion, systemic infection will be happened, so C. abortus is an important zoonosis pathogen. MacropHage infectivity potentiator(MIP) is a kind of lipoprotein which participate in chlamydia infection and cause inflammation reaction process as a immunodominant antigen.In this study, MIP is as a target protein to be studied, primers have been designed according to the published sequence of mip gene on the GenBank and PCR product has been gotten after amplification. The mip gene was inserted into prokaryotic expression vector pET30a(+) correctly after enzyme digestion, connection and transformation.The recombinant expression vector pET30a(+) has been transferred into E.coli BL21(DE) and a 27 k D molecular weight protein has been gotten after induced by ITPG. Its reactivity has been confirmed by SDS-PAGE experiment.The double antigens sandwich ELISA has been established with purified MIP protein as coated antigen and HRP labeled MIP as enzyme labeled antigen. The optimal concentration of coated antigen was 2.5 μg/mL and the dilution degree of serum was 1:100. The sensitivity of double antigens sandwich ELISA is higher than IHA, and the coincidence betweenthem was 89.3%. It has also no cross reaction with positive serums of mycoplasma, legionella pneumop Hila, foot-and-mouth disease. The inter batch and in batch of CV% were less than 11%. It indicated that the established double antigens sandwich ELISA has strong specificity, sensitivity and repeatability.The positive cell clones and limited dilution cloning has been done after Balb/c mouse were injected with purified recombinant MIP protein. Two hybridoma cell strains which can secret anti-MIP monoclonal antibodies stably has been established and named them as M1 and M3, respectively. The titer of these mAbs were 1:1600 by indirect ELISA; the characteristics of hybridoma was confirmed by chromosome identification assay and the relative affinity of them were 10 μg/mL; the western –blot test showed that the two strains of monoclonal antibodies could react specifically with the recombinant MIP protein; the subtype of IgG of two strains was identified by the mAb subclass detection kit and the results showed that all of them were IgG1; and the light chain of them was κ chain; the neutralization test showed that the mAbs can interrupt transmission of infection effectively through injection chicken embryo.In conclusion, the MIP protein has been expressed successfully and the double antigens sandwich ELISA has been established, which can detect C. abortus effectively. Furthermore, the anti-MIP monoclonal antibody has been gotten. All these finished work provide a foundation for the further research of correct detection and pathogenic mechanism of C. abortus...