Establishment An IELISA Of An Antibody Detection Method Based Against MSA-2c Of Babesia Bovis

The disease named Bovine babesiosis is a general name of blood protozoon disease.It is caused by kinds of genus Babesia.It is transmitted by ticks and parasitizes in the erythrocytes of cattle.The disease is usually characterized by fever,hemolytic anemia,hemoglobinuria,and enen death in severe cases.At present,microscope observation method is still the mainly method in detection in basement veterinarian of our country.The necessary terms that definite this illness is finding protozoon in blood film. This method is intuitive,but it also can easily caused misdiagnosis,so it make many difficulties to diagnose and prevent this disease.The purpose of my study for the above,to establish a specific and sensitive detection methods.It can detect this disease even in early infection cases.The plasmid pGEX-4T-2 that contain MSA-2c gene was tansformed into E.coli BL21(DE3).Electing the Clones that can express the protein in high level.The E.coli were induced by IPTG.And the protein was purified by glutathione sepharose 4B.The results of SDS-PAGE and Western-Blot show that the induced pGEX-4T-2/MSA-2c positive strain expressed a active recombined protein its molecular weight is 55KDa. ELISA show that the potency of antibody that was produced by immuning mouse using the recombined protein can reach 1:5,120,000.So this protein can be used as a stable tool of diagnose to Bovine babesiosis was proofed.The purified recombined Babesia Bovis MSA-2c protein was used as antigen,and HRP-labeled rabbit anti- cattle immunoglobulin G(IgG) conjugate was used as second antibody.The detection method of Babesia Bovis iELISA was established.The working conditions of iELISA were optimized by serial optimizing tests.The antigen concentration of coating is 2.5μg/mL,overnight at 4℃,serum and secondary antibody were attenuated as 1:20 and 1:1000 respectively.The optimal coating buffer,blocking solution and dilution solution were 0.05 M CB(pH9.6),3%BSA and PBST containing 0.1%BSA,respectively.The optimized reaction conditions of serum samples and the conjugate were both of 2h at 37℃,and the exposure time for TMB was about 20-25min at 37℃.The iELISA assay was performed by detecting MSA-2c antibodies of 95 cattle serum samples.The negative serum were attenuated as the best dilution. According to the statistics regular,the positive sample is OD₄₅₀nm＞(?)+3S.Calculated(?)=0.197,S=0.05. So the critical value between negtive and positive is 0.197+3×0.05=0.347.The distinctive regulation was established.When OD₄₅₀nm of samples≥0.347 is positive.When OD₄₅₀nm of samples＜0.347 is negtive. The cross-reactivity show that the recombined MSA-2c antigen can not react with other Piroplasmida serum.Its specific of this antigen is in high level.The detection method has high stability and reproducibility.The intraassay and interassay coefficient of variation canbe controlled less than 10%.Many detection of serum show that iELISA assay has good reproducibility,high specific,high sensitivity.