Simultaneous Detection of Forbidden Chemical Residues in Milk Using Dual-Label Time-Resolved Reverse Competitive Chemiluminescent Immunoassay Based on Amine Group Functionalized Surface

In this study, a sensitive dual-label time-resolved reverse competitive chemiluminescent immunoassay was developed for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) in milk. The strategy was performed based on the distinction of the kinetic characteristics of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) in chemiluminesecence (CL) systems and different orders of magnitude in HRP CL value for CAP and ALP CL value for CLE in the chemiluminescent immunoassay. Capture antibodies were covalently bound to the amine group functionalized chemiluminescent microtiter plate (MTP) for efficient binding of detection antibodies for the enzymes labeled CAP (HRP-CAP) and CLE (ALP-CLE). The CL signals were recorded at different time points by the automatic luminometers with significant distinction in the dynamic curves. When we considered the ALP CL value (about 10⁵) of CLE as background for HRP CL signal value (about 10⁷) of CAP, there was no interaction from ALP CL background of CLE and the differentiation of CAP and CLE can be easily achieved. The 50% inhibition concentration (IC₅₀) values of CAP and CLE in milk samples were 0.00501 µg L⁻¹ and 0.0128 µg L⁻¹, with the ranges from 0.0003 µg L⁻¹ to 0.0912 µg L⁻¹ and from 0.00385 µg L⁻¹ to 0.125 µg L⁻¹, respectively. The developed method is more sensitive and of less duration than the commercial ELISA kits, suitable for simultaneous screening of CAP and CLE.