Establishment And Preliminary Application Of Indirect ELISA For Detecting Antibody To Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG)is a highly transmissible pathogen of chronic respiratory disease and leads to cough,sneeze,tracheal rales and rhinitis in infected chicken,thus resulting in significant economic losses in the poultry industry.Enzyme-linked immunosorbent assay(ELISA)is a commonly serological detection method for the diagnosis of MG infection.At present,there is no commercially available ELISA test kit in China,and the imported diagnostic kit is too expensive to be suitable for large-scale application,which brings difficulty to control of MG in China.Therefore,the development of an indirect ELISA kits with independent intellectual property rights would provide an accurate,effective diagnostic tool for the epidemiological monitoring and eradication of MG in China.In the previous study in our laboratory,five heat shock and adhesin proteins including P25?P33?P37?P60 and P44 were initially identified.These membrane proteins were expressed and purified by prokaryotic expression system and analyzed by Western blot with MG positive serum.The results showed that the heat shock protein P60 had a strong immune response with MG positive serum,indicating that the protein had good immunogenicity.The rP60-iELISA for detection of MG antibody was established by using the purified rP60 as coating antigen.The optimum reaction condition for rP60-iELISA as follows: the concentration of coating antigen was 2.5?g/ml;the optimal dilution of serum sample was defined as 1:80 and incubation time was 60 min at 37°C;the sealing buffer was 5% skim milk and incubated at 37°C for 2h;the second antibody were 1:10000 diluted and incubated 30 min at 37°C;the colorimetric reaction time was 10min;the optimal cut-off point was 0.32.To assess whether rP60 had the potential to be developed as a diagnosis kit,technical indicators of MG kit such as sensitivity,specificity,accuracy,antibody duration,repeatability and stability were evaluated.The results were as follows:the sensitivity of this method was 98% and the minimum detection limit was 1280-fold dilution of positive serum;the specificity was 98% and rP60 had no cross-reaction with the positive serum of common respiratory pathogens;the CV of intra and inter assay were 1.54%~5.15% and 2.83%~5.98%,respectively;eight lives chickens with MG infection were tested by rP60-iELISA,a commercial kit,PCR and isolation,the rP60-iELISA method showed the same sensitivity as isolation;a total of 208 clinical serum samples were tested using rP60-iELISA and a commercial kit,and the agreement rate was 87.8%;the rP60-iELISA was more suitable for early detection than commercial kit;the kit was stable at 4°C for 8 months,and the CVs of the detection results of sensitivity and cross-reaction were less more than 10%.The above results showed that the established rP60-iELISA had good sensitivity,specificity,accuracy,repeatability and stability,which expected to provide an accurate detection tool for eradication and prevention of MG infection.