Development of an ultrasensitive indirect competitive chemiluminescent enzyme-linked immunoassay for the rapid detection of bacitracin in animal tissues and milk

In recent years, due to the unscientific and irrational use of bacitracin (BAC), the drug resistance of animal-derived bacteria has continued to increase, posing a threat to animal-derived food safety and public health safety. Therefore, there is an urgent need to develop a rapid and sensitive BAC assay. In this study, BAC-ovalbumin was used as the coated antigen, mouse monoclonal antibody was used as the detection antibody, and the working system was optimized. An indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for the detection of BAC was established. Under the optimal conditions, the half maximal inhibitory concentration of the method was 0.10 ng/mL, and the linear range of detection was 0.05–2.19 ng/mL. Limit of detection of the method was at least 500 times lower than the maximum residue limits. The sample spike recovery experiment revealed that the recovery rates for animal tissue and milk were 78.7 %–93.7 % and 81.3 %–96.7 %, respectively. The intra-assay and interassay coefficients of variation were all less than 15 %, with good precision and no cross-reaction with five bacitracin analogs. At the same time, HPLC also verified the reliability of the method. This method provides a convenient, accurate and rapid means of screening BAC residues in actual animal-derived foods. ● Screened hybridoma cell lines secreting anti-bacitracin monoclonal antibody have higher titer and sensitivity. ● The sensitivity of the indirect competitive chemiluminescent enzyme-linked immunoassay to detect bacitracin was 0.10 ng/mL. ● Real-world concentration of bacitracin in animal tissues and milk was successfully detected and verified.