A Novel Highly Sensitive Chemiluminescence Enzyme Immunoassay with Signal Enhancement Using Horseradish Peroxidase-Luminol-Hydrogen Peroxide Reaction for the Quantitation of Monoclonal Antibodies Used for Cancer Immunotherapy

Abstract

The development and validation of a novel enhanced chemiluminescence enzyme immunoassay (CLEIA) with excellent sensitivity for the quantification of monoclonal antibodies (mAbs) used for immunotherapy of cancer are described in this paper for the first time. The 96-microwell plates were used for the assay procedures, which involved the non-competitive binding reaction to a specific antigen. The immune complex of the antigen-mAb formed on the internal surface of the plate wells was quantified by a novel chemiluminescence (CL)-producing horseradish peroxidase (HRP) reaction. The reaction employed 4-(imidazol-1-yl)phenol (IMP) as a highly potent signal enhancer for the HRP-luminol–hydrogen peroxide (H2O2) CL reaction. The proposed CLEIA was developed for bevacizumab (BEV), as a representative example for mAbs. The CLEIA was validated in accordance with the immunoassay validation for bioanalysis standards, and all of the validation criteria were met. The assay’s limit of detection (LOD) and limit of quantitation (LOQ) were 9.3 and 28.2 pg mL−1, respectively, with a working dynamic range of 10–400 pg mL−1. The assay enables the accurate and precise quantitation of mAbs in human plasma samples without any interference from endogenous substances and/or plasma matrix. The novel CLEIA was compared in terms of dynamic range and sensitivity with other pre-validated enzyme-linked immunosorbent assay (ELISA) using HRP/colorimetric substrate as a detection system and the observed differences were explained. The CLEIA protocol’s ease of use, high throughput, and simplicity allows to analyze numerous samples in clinical settings. The proposed CLEIA has a significant benefit in the assessment of mAbs in clinical settings for the evaluation of their pharmacokinetics, pharmacodynamics, therapeutic drug monitoring, and refining their safety profiles, opening a new era for a better understanding of pharmacodynamics at the cellular level.