布鲁氏菌BP26合成肽ELISA鉴别诊断方法的建立OA

Establishment of a differential diagnostic ELISA method based on synthetic peptide derived from Brucella BP26 to distinguish antibodies induced by the Brucella gene deletion live vaccine(strain M5-90△26)from those caused by wild strain infection,and providing technical support for the differential diagnosis of Brucellosis.In this study,the Brucella BP26 synthetic peptide was used as the coating antigen.The optimal coating concentration of the BP26 synthetic peptide and the optimal dilution of serum were determined by checkerboard titration.Additionally,the optimal blocking buffer,reaction duration for serum,reaction duration for enzyme-labeled antibody,and cut-off value were systematically optimized.Following these steps,the sensitivity,specificity,reproducibility and differential diagnostic ability of the diagnostic method were evaluated.The results showed that the optimal coating concentration of the BP26 synthetic peptide was 1 μg/mL,the optimal dilution of the serum was 1∶20.The optimal blocking buffer was phosphate buffer containing 5％BSA.The optimal reaction duration for serum was 30 min,and 60 minutes for enzyme-labelled antibody.The samples were classified as positive when the OD450 ≥P(mean value of the positive control)×0.262,and negative when＜P(mean value of positive control)×0.262.The minimum detection limit of this method for the samples was 1∶1280 dilution.It demonstrated good specificity,showing no cross-reactivity with Clostridium perfringens,Escherichia coli,or Salmonella.The intra-batch coefficient of variation(CV)was 5.8％～8.4％,while the inter-batch CV was 6.6％～10.3％.In differential diagnostic test,the detection rate of BP26 positivity for 40 Brucella-positive serum was 77.5％,and the detection rate of BP26 positivity for 40 Brucella gene deletion live vaccine(strain M5-90 △26)immune serum was 0％.The established indirect ELISA method has the ability to differentiate Brucella gene deletion vaccine(strain M5-90△26)immunized serum from other Brucella positive serum.It is expected to provide a more effective differential diagnostic method for Brucella gene deletion vaccine(strain M5-90△26)immunized flocks.