Abstract: 【Objective】 The purpose of the test was to establish an indirect ELISA method for detection of antibodies to Mycoplasma bovis, and provide a method for testing the efficacy of inactivated vaccine against Mycoplasma bovis.【Method】 The prokaryotic expression plasmid pET-32a-P48 was constructed and transformed into E.coli BL21(DE3) Plyss competent cells to screen the positive plasmid and perform double digestion verification.The recombinant protein was induced to express with IPTG with a final concentration of 1 mmol/L, and purified with nickel column.The protein purity and molecular weight were verified by SDS-PAGE, and the specificity and reactogenicity of the recombinant P48 protein were detected by Western blotting.The polyclonal antibodies serum was prepared by immunizing rabbits with the recombinant P48 protein.An indirect ELISA antibody detection method for Mycoplasma bovis was established and the conditions of the method were optimized, the critical value was determined.The sensitivity, stability, specificity and coincidence rate of the established method with plate agglutination test were tested.【Result】 The molecular weight of P48 protein was 62.68 ku and its purity was over 90% by SDS-PAGE.Western blotting test results showed that P48 protein was only bound to the corresponding antibody and showed a single band, which showed good specificity and reactivity.The positive serum titer was 1:64 by agar diffusion method.The optimized results of the established indirect ELISA detection method were as follows:The antigen-coated concentration was 0.3 μg/mL, 1% BSA was blocked at 37℃ for 120 min, the serum to be tested was diluted at 1:1 280, incubated at 37℃ for 60 min, the secondary antibody dilution was 1:5 000, incubated at 37℃ for 60 min, and o-phenylenediamine was colored at room temperature for 10 min.D_{492 nm} value>0.3196 was positive, D_{492 nm} value<0.3101 was negative, 0.3101 ≤ D_{492 nm} value ≤ 0.3196 was suspicious.The results of sensitivity test showed that when the dilution of positive sample was 1:2¹⁸, the interpretation of positive serum was still positive.The stability test results showed that the coefficient of variation of the 96-well plates of 3 batches of P48 protein coated was less than 10%.There was no cross reaction with positive serum of Mannheimia haemolytica and Pasteurella.Compared with plate agglutination test, the coincidence rate of positive serum and negative serum was 100%(18/18) and 97.5%(39/40), respectively.【Conclusion】 The indirect ELISA antibody detection method for Mycoplasma bovis P48 established in this study had good sensitivity, specificity, and stability, providing a detection method for Mycoplasma bovis antibody detection and Mycoplasma bovis vaccine efficacy detection

Key words: Mycoplasma bovis; P48 protein; indirect ELISA