Preparation of monoclonal antibody against azithromycin and establishment of immunoassay for azithromycin

Objective To prepare mouse monoclonal antibody (mAb) with haptens of azithromycin (AZM) and characterize its features. Methods The immunogen AZM-BSA and coating antigen AZM-OVA were synthesized by coupling AZM to the bovine serum albumin (BSA) and ovalbumin (OVA) with the activated ester method. Hybridoma cell line 50D10 was obtained by fusing mouse myeloma cells Sp2/0 with splenocytes from the BALB/c mice immunized with AZM-BSA. The cell line was then injected into BALB/c mice to prepare mAb against AZM, which was detected by ELISA using this mAb. Results The titer of AZM mAb was 1:4.0×105, which belonged to IgG1 type. The sensitivity and IC50 of the indirect competitive ELISA was 0.287 μg/L and 1.33 μg/L, respectively. The cross-reactivity rates of the mAb with erythromycin, roxithromycin, doramectin, clarithromycin, tilmicosin and tylosin were less than 0.1%. The recovery rates of AZM added into milk ranged from 79.6% to 104.1% with the coefficients of variation between 16.4% and 27.3%. Conclusion AZM mAb with high sensitivity and specificity has been prepared, which lays a foundation for establishing a method to rapidly detect AZM.