Time-resolved And Spatial-resolved Chemiluminescence Strategy For Multiplexed Immunoassay

As a promising analytical technique with high sensitivity and high selectivity, chemiluminescence immunoassay (CLIA) is widely applied in various areas such as clinical diagnosis, environmental monitoring and food safety. With the development of immunoassay technique, multiplexed immunoassay (MIA) has been increasingly necessary to quantitate several components in complex biological samples. For instance, to monitor effectively several β2-agonists used as "lean meat agents", it is essential to develop MIA to replace the traditional parallel single-analyte immunoassay. MIA has already attracted growing attention due to its high sample throughput, short analytical time, low sample consumption, and low assay cost. Thus, considerable effort has been paid to the development of various MIA strategies.Up to now, most reported MIA methods can be classified into two dominant modes. The first one is the multilabel mode which is performed with different labels, and the second one is the spatial-resolved mode which utilizes a universal probe to tag all analytes. Herein, the two MIA methods respectively based on the multilabel mode and the spatial-resolved mode are presented.Part1Time-resolved chemiluminescence (CL) strategy for multiplexed immunoassayA novel time-resolved 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 (CLE) and ractopamine (RAC) 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 CLE and RAC were in turn detected after0.6s and25min 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 CLE and RAC were both1.0-500ng/mL, with detection limits of0.50ng/mL (S/N=2). The results for real sample analysis demonstrated that this study could provide a simple, reliable and low-cost approach toward multiplexed immunoassay.Part2Development of a multianalyte lateral-flow immunochromatographic technique based on spatial-resolved CL strategyIn this paper, a multianalyte lateral-flow immunochromatographic assay based on spatial-resolved CL strategy was developed for the rapid simultaneous determination of RAC and salbutamol (SAL). First, BSA-RAC and BSA-SAL were respectively immobilized on the different areas of nitrocellulose membrane to form two test zones. HRP was used as signal probe to label the two tracer antibodies. After the competitive immunoreactions, the CL signals from the two test zones were determined. The linear ranges for RAC and SAL were0.50-40and0.10-50ng/mL, with the detection limits for RAC and SAL were170and33pg/mL (S/N=3), respectively. This proposed lateral-flow immunochromatographic assay has a series of advantages such as high sensitivity, simple manipulation, low cost et al. Thus, it shows great promise in food safety.