Background: Cetuximab (CET) is a monoclonal antibody that inhibits Epidermal Growth
Factor Receptor (EGFR) used for immunotherapy of different types of cancer. Additionally, CET used
in combination therapy, potentiates the effects of chemotherapy and radiation therapy in eradicating
well-established tumors. Recently, a combination of CET and newly developed chemotherapeutic candidate
drugs are being investigated for use as new-generation chemotherapy.
Introduction: Due to the increasing importance of this class of biopharmaceuticals, our dynamic field
of research has arisen around the establishment of an immunoassay method with a highly specific and
sensitive to quantify CET at low concentration levels in human plasma. This study describes the development
and full validation of a new enzyme-linked immunosorbent assay (ELISA) with high sensitivity
and selectivity for bioanalysis of CET.
Methods: The assay involved the non-competitive binding reaction of CET to its specific antigen (human
epidermal growth factor protein; EGFR) followed by a chromogenic enzyme reaction for immune
detection of the CET- EGFR complex and color development. Technically, CET was captured by EGFR
antigen protein immobilized onto a 96-well assay plate. The CET- EGFR complex formed onto the
plate wells was 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
Results: 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.
The assay Limit Of Detection (LOD) was 1.5 ng/mL and the effective working dynamic range was 5-
6250 ng/mL. The accuracy and precision of the assay were proved.
Conclusion: We have developed and validated a highly sensitive and selective ELISA for quantitation
of CET in plasma samples. 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 paranitrophenyl
phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme.
An analyst can process a batch of ~ 200 samples, in triplicate, per day. The proposed ELISA method for
CET shows acceptable precision and, accuracy and adequate sensitivity to contribute to study its pharmacokinetic
(PK), Pharmacodynamic (PD), Therapeutic Drug Monitoring (TDM) and appears to be
suitable for employment in all clinical laboratories.