布鲁氏菌抗体荧光微球免疫层析检测方法的建立OA

To develop a rapid immunochromatographic method for detecting Brucella antibodies in sheep serum,fluorescent microspheres was used as labeling carriers to conjugate with recombinant Brucella outer membrane protein BP26.BP26 protein and its monoclonal antibody 1D10 were respectively dispensed onto the nitrocellulose membrane as the test line and control line.After optimizing the reaction conditions,the fluorescent microsphere test strip was assembled and its specificity,sensitivity,and repeatability were evaluated.The performance of this method was also compared with a commercial Brucella competitive enzyme linked immunosorbent assay(cELISA)antibody detection kit using 184 clinical sheep serum samples.The results showed that the optimal reconstitution dilution of the fluorescent microsphere conjugate was 200 times,the optimal coating concentration of the test line was 0.6 mg/mL,and the optimal reaction time was 15 minutes.The test strip exhibited no cross-reactivity with positive sera against Mannheimia haemolytica,Salmonella,Yersinia enterocolitica,peste des petits ruminants virus,and foot-and-mouth disease virus.The maximum dilution ratio for detecting Brucella-positive reference serum was 1∶800.Both intra-batch and inter-batch coefficients of variation were below 10%.The overall agreement rate between the test strip and the cELISA kit for clinical samples reached 92.4%.These results indicate that the fluorescent microsphere immunochromatographic strip developed in this study has high specificity,strong sensitivity,stability,rapidity,and low cost.It is suitable for rapid clinical screening and epidemiological investigation of Brucella antibodies.