This study describes, for the first time, the development and validation of a novel ultrasensitive chemiluminescence enzyme immunoassay (CLEIA) for the quantification of atezolizumab (ATZ), a monoclonal antibody approved by the FDA for treatment of different types of cancer. The assay involved the non-competitive binding of ATZ to its specific antigen (PD-L1 protein). The immune complex of PD-L1/ATZ formed on the internal surface of the plate wells was quantified by a novel chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed a highly efficient CL enhancer for the HRP-luminol-hydrogen peroxide reaction which was 4-(imidazol-1-yl)phenol. The conditions of the CLEIA and its detection system were refined, and the optimum procedures were established. The CLEIA was validated in accordance with the guidelines of immunoassay validation for bioanalysis, and all the validation criteria were acceptable. The assay's limit of detection and limit of quantitation were 12.5 and 37.5 pg mL-1, respectively, with a working dynamic range of 25-800 pg mL-1. The assay enables the accurate and precise quantitation of ATZ in human plasma samples without any interferences from endogenous substances and/or the plasma matrix. The results of the proposed CLEIA were favourably comparable with those of a pre-validated enzyme-linked immunosorbent assay using a colorimetric detection system. The CLEIA is characterized by simple and high throughput features. The CLEIA is superior to the existing analytical methodologies for ATZ. The proposed CLEIA has a great value in the quantitation of ATZ in clinical settings for assessment of its pharmacokinetics, therapeutic drug monitoring, and refining the safety profile.
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