Immunotechniques for the Group Determination of Macrolide Antibiotics Traces in the Environment Using a Volume-Mediated Sensitivity Enhancement Strategy

Macrolide antibiotics, effective antimicrobial agents, are intensively used in human and veterinary medicine as well as in agriculture and therefore are found all over the world as environmental pollutants, harming sensitive eco-community organisms and provoking the selection of resistant forms. A novel azithromycin derivative was synthesized and as a rationally designed hapten conjugate ensured group immunorecognition of 6 major macrolide representatives (105-41%), erythromycin, erythromycin ethylsuccinate, clarithromycin, roxithromycin, azithromycin and dirithromycin in competitive immunoassay based on anti-clarithromycin antibodies. The heterologous hapten-based ELISA format simultaneously contributed to a 5-fold increase in sensitivity (ERY IC50 = 0.04 ng/mL). However, for the detection of trace macrolides in environmental samples, an underexploited in immunoassay field strategy was proposed in the present study to significantly improve the detectability of analytes. Unlike most approaches, it does not require special enhancers/amplifiers or additional concentration/extraction procedures, but only involves a larger volume of test samples. Gradual volume increase of samples (from 0.025 to 10 mL) analyzed in direct competitive ELISA, immunobeads, and immunofiltration assay formats based on the same reagents resulted in a significant improvement (more than 50-fold) in assay sensitivity and detection limit up to 5 and 1 pg/mL, respectively. The suitability of the test for detecting macrolide contamination of natural water was confirmed by recovery of macrolides from spiked blank samples (71.7-141.3%). A series of natural water samples from Lake Onega and its influents near Petrozavodsk were analyzed during a 2022-2023 using both the developed immunoassay and HPLC-MS/MS and revealed no macrolide antibiotic contamination.