Cetuximab (Erbitux®) is a chimeric IgG1 monoclonal antibody that binds the extra-cellular domain of the epidermal growth factor receptor (EGFR). It is a 152-kDa molecule composed of four polypeptide chains: two identical heavy chains and two identical light chains, consisting of 449 and 214 amino acids, respectively, bound by covalent and non-covalent bonds. The bond with EGFR is characterized by a higher affinity than either endogenous ligand, as epidermal growth factor (EGF), or transforming growth factor alpha. This binding inhibits activation of the receptor tyrosine kinase and the associated downstream signaling that includes the mitogenactivated protein kinase, phosphoinositide 3-kinase/Akt and the Janus kinases/ signal transducers and activator of transcription (Stat) pathways. Furthermore Cetuximab induces antibody-mediated receptor dimerization, internalization and degradation leading to receptor down-regulation. In addition, it exhibits antibody-dependent cellular cytotoxicity that could contribute to its antitumor effect.
PRINCIPLE OF THE ASSAY
This assay employs the sandwich enzyme immunoassay technique. Anti- Cetuximab is coated onto a 96 well microplate. Calibrator, quality control samples (if desired) and test samples are pipetted into the appropriate wells. Cetuximab present in biological matrices is bound by the immobilized anti- Cetuximab antibody. After washing away any unbound substances, enzyme linked anti- Cetuximab antibody is added to the wells. This antibody is developed and purified specifically against truncated Erbitux® (domain residing in Fc portion of the Erbitux® molecule). The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of Cetuximab present in test samples. The color development is stopped and the intensity of the color is measured.