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Group determination of 14-membered macrolide antibiotics and azithromycin using antibodies against common epitopes
Inna Galvidis1, Gennady Lapa2, Maksim Burkin1
1Department of Hybridomas, Mechnikov Research Institute for Vaccines and Sera, Russian Academy of Medical Sciences, Moscow 105064, Russia.
Abstract:
Erythromycin (ERY), clarithromycin (CLA), roxithromycin (ROX), and azithromycin (AZI) are macrolide antibiotics widely used in livestock and human medicine. Therefore, they are frequently found as pollutants in environmental water. A method based on indirect competitive enzyme-linked immunosorbent assay (ELISA) for group determination of these macrolides in foodstuffs, human biofluids, and water was developed. Carboxymethyloxime of clarithromycin (CMO-CLA) was synthesized and conjugated to bovine serum albumin (BSA) and gelatin to prepare immunogen and coating antigen with advantageous presentation of target epitopes, l-cladinose and d-desosamine, common for these analytes. Antibodies generated in rabbits were capable of recognizing ERY, CLA, and ROX as a group (100-150%), and AZI (12%) and did not cross-react with ERY degradants, which lack antibiotic activity. Assay displayed sensitivity of determination of 14-membered macrolides (IC50=0.13-0.2ng/ml) and low limit of detection (LOD) that was achieved at 0.02 to 0.03ng/ml. It allowed performing analysis of milk, muscle, eggs, bovine serum, water, human serum and urine, and avoiding matrix effect without special pretreatment using simple dilution with assay buffer. For 15-membered macrolide AZI, the corresponding characteristics were IC50=1.6ng/ml and LOD=0.14ng/ml. The recoveries of veterinary and human medicine macrolides from corresponding matrices were validated and found to be satisfactory.
Insights
A new enzyme-linked immunosorbent assay (ELISA) can detect multiple macrolide antibiotics, including erythromycin and clarithromycin, in various samples like water and food. This method offers high sensitivity for environmental and biological monitoring of these common pollutants.
Area of Science:
- Environmental Chemistry
- Analytical Chemistry
- Immunochemistry
Background:
- Macrolide antibiotics (erythromycin, clarithromycin, roxithromycin, azithromycin) are extensively used in human and veterinary medicine.
- These widely used antibiotics are frequently detected as environmental pollutants, particularly in water bodies.
Purpose of the Study:
- To develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the simultaneous determination of four major macrolide antibiotics.
- To enable the detection of these macrolides in diverse matrices including foodstuffs, human biofluids, and environmental water samples.
Main Methods:
- Development of an indirect competitive ELISA utilizing a synthesized carboxymethyloxime of clarithromycin (CMO-CLA) conjugate.
- Antibodies were generated in rabbits against the immunogen and coating antigen, designed for optimal epitope presentation.
- The assay was optimized for sensitivity, specificity, and matrix effect mitigation through simple dilution.
Main Results:
- The developed ELISA demonstrated high sensitivity for 14-membered macrolides (IC50 = 0.13-0.2 ng/mL, LOD = 0.02-0.03 ng/mL).
- Azithromycin (15-membered macrolide) showed distinct assay characteristics (IC50 = 1.6 ng/mL, LOD = 0.14 ng/mL).
- The assay successfully analyzed various matrices (milk, muscle, eggs, serum, urine, water) without significant matrix effects and showed satisfactory recovery rates.
Conclusions:
- The developed group-specific ELISA provides a reliable method for detecting common macrolide antibiotics in environmental and biological samples.
- This assay facilitates the monitoring of macrolide pollution and exposure in diverse settings.
- The method's sensitivity and ability to handle complex matrices without extensive pretreatment make it a valuable tool for routine analysis.
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