关键词: 胞内劳森菌, 单克隆抗体, 夹心ELISA, 抗原定量

Abstract:

【Objective】This study established a sandwich ELISA method for detecting Lawsonia intracellularis (L. intracellularis), which could be used to measure antigen content in the development of porcine proliferative enteropathy (PPE) inactivated vaccines.【Method】L. intracellularis was used as the immunogen, and polyclonal antibody against L. intracellularis was obtained by immunizing New Zealand rabbit. Additionally, 6-week-old BALB/c mice were immunized to prepare monoclonal antibodies against L. intracellularis by hybridoma cell technique. The antibodies were identified by Western Blot, indirect immunofluorescence (IFA) and immunoperoxidase monolayer assay (IPMA). A sandwich ELISA method was established for detecting L. intracellularis, using monoclonal antibody as capture antibody and polyclonal antibody as detection antibody. the optimized reaction conditions and the specificity, sensitivity and reproducibility of the method were evaluated. The optimized sandwich ELISA was used to quantify inactivated L. intracellularis.【Result】The polyclonal antibody titer reached 1﹕409 600. After three rounds of subcloning, three positive hybridoma cell lines were screened, named 1B7, 3C7, and 4F10, respectively, and the titer of ascites were all 1﹕204 800. All three monoclonal antibodies had specific reactions with L. intracellularis and showed no cross-reactivity with E.coli O157:H7, S. choleraesuis, and S. typhimurium. The optimal conditions for the sandwich ELISA were as follows: capture antibody was 4F10; working concentrations for capture and detection antibodies were 1.25 and 0.625 μg·mL^-1, respectively. The coating solution was phosphate buffer, and the coating condition was 4 ℃ for 14 h. Blocking solution was 1% gelatin and blocking condition was 37 ℃ for 1.5 h. The reaction condition of antigen was 37 ℃ for 1 h, and the reaction condition for detection antibody was 37 ℃ for 0.5 h. HRP-antibody dilution was 1﹕16 000 and reaction condition of HRP-antibody was 37 ℃ for 1.5 h. The developing time was 10 min at room temperature. This method was negative for other common intestinal pathogens, with a minimum detection limit of 1×10⁵ L. intracellularis/mL. The coefficient of variation for intra and inter assay were less than 10%. A linear relationship was observed between L. intracellularis concentration and P/N values in the range of 1×10⁵ - 1×10⁸, with the standard equation : y=2.7349x-11.643 (R²=0.9966). This method was used to quantify the antigen content of PPE inactivated vaccines in our laboratory. The quantitative results indicated that the antigen content remained basically unchanged before and after inactivation, with two groups of vaccine samples demonstrating good intra-assay reproducibility. The results of 120 clinical fecal samples of swine showed that 62 positive samples and 58 negative samples were detected using the sandwich ELISA method; 67 positive samples and 53 negative samples were detected using nested PCR method. The positive coincidence rate of the two methods was 86.6% and the negative coincidence rate was 92.5%, the total coincidence rate of the two methods was 89.2%, and the kappa value was 0.78.【Conclusion】Monoclonal antibody and polyclonal antibody against L.intracellularis were successfully prepared, and a sandwich ELISA method for detecting L.intracellularis was successfully established and optimized. This method had good specificity and reproducibility, with a detection limit of 1×10⁵ mL^-1 for L.intracellularis, which could be used for the quantification of antigen content in the production of PPE inactivated vaccine and detection of PPE clinical samples.

Key words: Lawsonia intracellularis, monoclonal antibody, sandwich ELISA, antigen quantification