Preparation of a highly specific and sensitive monoclonal antibody against tilmicosin and its application in lateral flow immunoassay

To reduce the false positive results caused by cross reactivity of the antibodies with other structural analogues, it is crucial to prepare a high specificity and sensitivity antibody against target for developing an accurate immunoassay. In this study, tilmicosin (TM) was selected as a model molecule. Firstly, two-dimensional similarity, electrostatic potential energy, mulliken atomic charges and overlapping of different haptens with TM were calculated using Gaussian 09W and Discovery studio, and the newly designed TM-HS was selected as the optimal hapten. Furthermore, a monoclonal antibody (mAb 12C8) was produced with the half maximal inhibitory concentration (IC50) of 0.36 ng/mL, and negligible cross-reactivity (CR) with other antibiotics. Finally, a lateral flow immunoassay (LFA) for the detection of TM based on amorphous carbon nanoparticles (ACNPs) labeled mAb 12C8 was developed by the reflectance value under natural light. The recoveries of TM ranged from 83.18% to 103.25% with a coefficient of variation (CV) < 12.47%. The results showed that the cut-off value of TM in milk samples was 1 ng/mL, and the limits of detection (LODs) for chicken muscle, bovine muscle, porcine muscle and porcine liver samples were 5.23, 5.98, 6.85 and 7.31 μg/kg, respectively. In addition, 40 real samples were tested by the LFA, and the detection results were consisted with that of high-performance liquid chromatography-UV detector (HPLC–UV). Those results indicated that the developed LFA is an accurate and useful tool for on-site screening of TM in milk and animal tissues.