Screening Of Specific Diagnostic Targets For M.ovipneumoniae And Establishment Of ELISA Antibody Detection Methods

Mycoplasma ovipneumoniae(Mycoplasma ovipneumoniae,Mo)is one of the causes of pneumonia.The clinical manifestations of the affected sheep are wheezing,cough,fever,progressive wasting,serous and cellulosic inflammation of the chest and pleura,and the infected sheep are easily secondary to infection with other pathogens.Vaccination and timely diagnosis are the main means to prevent and control the pathogen.The existing commercial kits are still unable to make the differential diagnosis of Mo,causing trouble to the timely diagnosis of Mo.In this paper,Mo antigen targets with conservative,specific and high confidence values were selected by Pull down and LC-MS analysis,prokaryotes were expressed and purified,analyzed purity and reacogenicity by SDS-PAGE and Western-blot,immunized mice,established the indirect ELISA method based on candidate target protein,and evaluated the sensitivity,specificity,repeatability,and compliance with commercialization.The results showed that the candidate diagnostic antigens for Mo based on mass spectrometry were Hsp 70,pdhD,pdhC and Enolase;Hsp 70 and pdhD had good immunogenicity and reacogenicity;the indirect ELISA method based on Hsp 70 and pdhD showed good specificity,sensitivity,repeatability and compliance rate,but Hsp 70 protein stability was better.Mouse source-specific monoclonal antibodies were prepared using the selected Hsp 70 protein.Positive hybridoma cell lines were screened by indirect ELISA,Balb /c mice,ascites was prepared and mAb was purified;the purity of mAb was determined by SDS-PAGE,and the specificity of mAb was determined by Western-blot;the blocking ELISA method was established to evaluate the sensitivity,specificity,reproducibility and conducibility.The results show that 9 of the 30 hybridoma cells positive cells,of which the supernatant of 1H5 hybridoma cell line not only has high blocking rate,and the mab has high concentration,purity and specificity,the blocking ELISA method based on the mab has good specificity,sensitivity,repeatability and compliance rate.In conclusion,the indirect ELISA with Mo serum antibodies was established with pdhD and Hsp 70 as target antigen and the blocking ELISA with Hsp 70 protein and HRP-labeled HRP-1H5 mAb.The above ELISA technique can specifically detect Mo serum antibodies(no cross-reaction with positive sera of goat pneumonia,goat and filamentous species),which laid a foundation for further development of Mo ELISA antibody detection kit.