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Development of a monoclonal antibody based-ELISA for the detection of chloramphenicol in shrimp, feed and milk samples and validation by LC-MS/MS coupled with immunoaffinity clean-up

Liu, Chang, Deng, Diandian, Xu, Di, Wu, Kang, Yang, Hong, Zhao, Kang, Li, Jianguo, Deng, Anping
Analytical methods 2019 v.11 no.4 pp. 507-516
antibiotics, antigens, cell fusion, chemical bonding, cross reaction, detection limit, enzyme-linked immunosorbent assay, florfenicol, gels, hybridomas, inhibitory concentration 50, liquid chromatography, mice, milk, mixing, monoclonal antibodies, shrimp, solid phase extraction, splenocytes, tandem mass spectrometry
In this study, a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) for the determination of chloramphenicol (CAP) in shrimp, feed and milk samples was developed and validated by LC-MS/MS coupled with immunoaffinity clean-up (LC-MS/MS-IAC). Three immunogens were synthesized and used to immunize mice. Spleen cells from the immunized mice with the highest titer were selected for cell fusion. Two lines of hybridoma cells (1B1 and 2D2) secreting specific antibodies against CAP were successfully screened. Under optimal conditions, the values of the 50% inhibitory concentration (IC₅₀) and limit of detection (LOD) of the ELISA in phosphate-buffered saline (PBS) based on 2D2 mAb for the detection of CAP were 0.46 ng mL⁻¹ and 0.06 ng mL⁻¹, respectively. There was no cross-reactivity (CR) of the ELISA with other antibiotics (florfenicol, thiamphenicol, etc.). Shrimp, feed and milk samples were spiked with different contents of CAP and detected by ELISA. The recoveries and coefficients of variation (CVs) of the ELISA for CAP from three spiked samples were 80.6–116.0% and 5.5–15.4% (n = 3), 96.2–111.0% and 7.0–13.5% (n = 3), and 79.7–109.8% and 5.8–13.3% (n = 3), respectively. As the CAP concentrations in spiked samples measured by LC-MS/MS coupled with solid phase extraction (LC-MS/MS-SPE) were significantly lower than those measured by ELISA, we established an immunoaffinity clean-up (IAC) for CAP by covalently immobilizing our mAb against CAP on the CNBr-activated Sepharose-4B gel and directly mixing the mAb-immobilized gel with the spiked samples sufficiently. The recoveries of CAP from the spiked samples measured by LC-MS/MS-IAC were 78.6–106.7% with the CV less than 11.1% (n = 3).