[Preparation of tiamulin artificial complete antigen and development of its monoclonal antibody]

Objective To develop high-sensitivity and high-specificity monoclonal antibody against tiamulin (TML). Methods Using oximation and carbodiimide method, TML was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare the immunogen TML-BSA and coating antigen TML-OVA, respectively. BALB/c mice were immunized with TML-BSA. The anti-TML monoclonal antibody hybridoma cell lines were screened by indirect ELISA after cell fusion. The ascites were prepared by in vivo induction method. The monoclonal antibody was purified from ascites by caprylic acid-ammonium sulfate method and identified by SDS-PAGE. Results The hybridoma cell lines of 3B3 and 4A7 were successfully obtained. Their titers of cell supernatant were 1:1600 and 1:6400, respectively. After purification, the titer of monoclonal antibody 4A7 was 5.12×10⁵. The half inhibitory concentration (IC₅₀) of 4A7 was 0.049 ng/mL. The affinity constant Kaff of 4A7 was 1.47×10⁹ L/mol. The cross-reaction rate of 4A7 was 2.7% with analog retapamulin, whereas no cross-reaction was detected with other antibiotics such as valnemulin and fleroxacin. Conclusion Anti-TML monoclonal antibody has been successfully developed.