Multiplexed Immunoassay Detection Of Immunoglobulin By A Reaction Kinetics-resolved Chemiluminescence Strategy

Chemiluminescent immunoassay (CLIA) is a promising non-radiolabeled immunoassay technique with highly sensitive and high specificity. It opens up a new pathway for pharmaceutical analysis, bioanalysis and clinical chemistry.CLIA is widely applied in various areas such as clinical diagnosis, environmental monitoring and food safety because of its advantages such as no extra light, high sensitivity, high specificity, wide linear range, no radioactive contamination, simple instrumentation.There is increasing interest in developing multiplexed immunoassay (MIA) that can substitute parallel single-annalyte immnunoassays in clinical diagnosis, environmental monitoring, and biodefense applications. A MIA shows some unique advantages, such as less sample consumption, shorter assay time, minimized repetitions of tedious procedures, and lower cost per test, in comparison with the conventional parallel single analyte detection. Nowadays, spatial-resolved mode and multi-label mode have been widely utilized in a MIA. Considerable effort is mainly focused on the development of multi-label-based multianalyte detection methods. Herein, the MIA method based on the multi-label mode is presented.A multiplexed immunoassay method was proposed for the sequential detection of two proteins in a single run based on a novel chemiluminescence (CL) reaction kinetics-resolved strategy. This method was established using acridinium ester (AE) and alkaline phosphatase (ALP) as the signal probes due to the significant difference in their CL reaction kinetics characteristics. Mouse IgG (MIgG) and mouse IgM (MIgM) were detected as the model analytes with a competitive immunoassay format. AE and ALP were used to tag goat anti-mouse IgG and rabbit anti-mouse IgM, respectively, to form two immunocomplexes. The two CL reactions with flash type and glow type kinetics characteristics were triggered simultaneously by adding the coreactants, then the CL signals from the two reactions were recorded after 0.2 s and 500 s of the reaction triggering, respectively. The multiplexed CL immunoassay provided a wide range of 0.50-200 ng mL-1, with a low detection limit of 0.16 ng mL-1 (S/N= 3) for both MIgG and MIgM. Additionally, no obvious signal overlap was observed in the multiplexed immunoassay. The proposed method was successfully applied for the detection of MIgG and MIgM levels in mouse serums, and the results were in good agreement with those from the reference ELISA method. This proposed method shows a series of advantages such as high sensitivity, ideal selectivity, simple manipulation. Thus, it opens up a new pathway for drug screening, food safety, environment monitoring and clinical diagnosis.