The Detection Of Antibody Agaist Of Mycoplasma Bovis Colloidal Gold Immunochromatographic Strip Development And Preliminary Application

Mycoplasma bovis（M.bovis） is a small cell wallless organism belonging to the class Mycoplasma,order Mycoplasma and genus Mycoplasma,which can cause respiratory diseases,mastitis,otitis media,conjunctivitis,meningitis and arthritis in cattle.The outbreak and prevalence of mycoplasma bovis disease in different regions of China have caused serious economic losses to the aquaculture industry.Early diagnosis and early prevention are the key technical links of prevention and treatment,but there is no commercial vaccine for Mycoplasma bovis at present,and the therapeutic effect of antibiotics is limited.At present,common PCR,fluorescence quantitative PCR,ELISA antibody detection and other laboratory methods have been used for laboratory diagnosis of Mycoplasma bovis,but portable rapid on-site diagnosis method can really meet the needs of field diagnosis.In this study,based on membrane proteins P48 and P80 of Mycoplasma bovis,an immunocolloidal gold chromatographic strip was established that could be applied in field detection of Mycoplasma bovis antibody.Firstly,p ET32a（+）-p48 and p ET32a（+）-p80 recombinant expression plasmids were transformed into BL21（DE3）competent cells,and positive monoclonal colonies were selected for induction and expression and purification.The purity,reactivity and concentration of purified proteins were detected by SDS-PAGE,Western-blotting and BCA kits,respectively.The results showed that P80 and P48 had good reactivity with the serum of high immunity Mycoplasma bovis.The purity and concentration of the proteins expressed were above 90%,which met the requirements of the subsequent development of the test strip.Colloidal gold particles at 35 nm were prepared by trisodium citrate reduction method.According to its detection methods,it was confirmed that the prepared gold particles met the labeling requirements.The optimal labeling p H value and labeling protein amount of recombinant protein P48,P80 and chicken Ig Y antibody labeled on colloidal gold were screened.It was determined that the optimal labeling p H value could be reached when 0.1 mo L/L K2CO3 was added into 1m L gold solution at 10μL,16μL and 12μL,respectively.the optimal labeling protein amount could be reached when P48,P80 and chicken Ig Y were added in 14μg,10μg and 6μg into 1m L gold solution,respectively.The gold-labeled protein and gold-labeled antibody solution were gradiently diluted to determine the optimal concentration of D₅₂₃=20.0 and D₅₂₃=5.0,respectively.Gradient concentration of protein A+G（test line）and goat anti-chicken Ig Y antibody（quality control line）were used to detect the standard negative and positive serum,and the optimal concentration was determined to be 1 mg/m L.The minimum detection limit of standard positive serum for Mycoplasma bovis was 10^-6,and the results of four positive samples such as Mycoplasma Ricchii were all negative,indicating that the strip had good sensitivity and specificity.The gold standard solution was sprayed on the glass fiber at the rate of 2μL/cm,and protein A+G and anti-chicken Ig Y antibody were sprayed on the nitric acid fiber film at the rate of 1μL/cm.Then the optimized strip was assembled.The earliest detection time of Mycoplasma bovis antibody by the test strip was indeed set at the14th day.The 46 positive sera and 77 negative sera prepared in the laboratory and 81 positive sera and61 negative sera in the clinical sample bank were tested and evaluated.Compared with the test results of commercial kits,the positive coincidence rates were 100%and 90.12%,and the negative coincidence rates were 89.61%and 98.36%,respectively.In conclusion,the immunocolloidal gold chromatographic strip developed in this experiment has the characteristics of short detection time,simple operation,easy to preserve,low production cost and easy to carry,etc.,which can be used for rapid detection of Mycoplasma bovis antibody in the field.