Time-resolved chemiluminescence strategy for multiplexed immunoassay of clenbuterol and ractopamine

A novel time-resolved chemiluminescence (CL) strategy was proposed for immunoassay of multiple analytes in a single run. The strategy was performed based on the distinction of the kinetic characteristics of different CL reaction systems, which allowed detection of multiple analytes in different time windows. The strategy was evaluated by using clenbuterol and ractopamine as the model analytes. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the two antigens due to their very different CL kinetic characteristics. After the competitive immunoreactions, the two CL signals were simultaneously triggered by adding the CL coreactants. Then the signals for clenbuterol and ractopamine were in turn detected after 0.6 s and 25 min of the reaction triggering. Due to the distinguishable detection time windows for HRP and ALP, the cross-talk resulting from the mixed CL reaction systems was effectively avoided, which was frequently encountered in some other multiplexed immunoassays based on multi-label modes. The linear ranges for clenbuterol and ractopamine were both 1.0-500 ng/mL, with detection limits of 0.50 ng/mL (S/N=2). The results for real sample analysis demonstrated that this study could provide a simple, low-cost and fast approach toward multiplexed immunoassay.