Two novel chemiluminescence immunoassays using the horseradish peroxidase (HRP)–luminol chemiluminescence system and two different enzymatic systems—HRP and alkaline phosphatase (ALP) chemiluminescence systems—were established for simultaneous detection of chloramphenicol (CAP) and clenbuterol (CLE) residues in milk. Milk samples were detected utilizing the two hybrid chemiluminescence immunoassays, with limits of detection (LODs) of 0.006 μg L−1 for CAP and 0.02 μg L−1 for CLE (Scheme 1A), 0.008 μg L−1 for CAP and 0.023 μg L−1 for CLE (Scheme 1B), respectively. The recoveries for CAP and CLE in individual and co-spiked samples were found to be between 80.0% and 96.7%. The applicability of the proposed method has been validated by determining CAP and CLE in field milk samples with satisfactory results. Thereafter, the developed hybrid chemiluminescence immunoassays can be used as reliable, rapid, and cost effective screening techniques for the simultaneous determination of CAP and CLE residues in milk.
