This article explores the development, full optimization and validation study of a highly sensitive and selective ELISA for determination of cetuximab (CET) in human plasma samples. CET was captured by specific epidermal growth factor receptor (EGFR) antigen protein immobilized onto a 96-well assay plate. The CET- EGFR complex formed onto the plate wells were quantified, for the first time, using alkaline phosphatase enzyme labeled anti-human IgG (ALP-IgG) and para-nitrophenyl phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme. The optimum conditions for conducting the proposed ELISA were established and its analytical performance was evaluated as per the guidelines for the validation of immunoassays for bioanalysis of therapeutic monoclonal antibody. The assay limit of detection was 0.0015 μg/mL and the effective working dynamic range was 0.005-6.25 μg/mL. The accuracy and precision of the assay were proved. The present assay with high throughput analysis very easy to perform in a 96-well plate and permits an operator to analyze a batch of ~ 200 samples, in triplicate. This facilitates the processing of a large number of samples in a clinical setting. The developed method eliminates the need for pretreatment of plasma samples by affinity chromatography or other sophisticated equipment. ELISA for the CET is expected to contribute in studying its PK, PD and TDM. The used reagents are commercially available, and the color product can be easily measured by inexpensive equipment available in biological laboratories.
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Ibrahim A. Darwish, Mona M. Al-Shehri and Manal A. El-Gendy*
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.