Cetuximab (Erbitux®) is a chimeric IgG1 monoclonal antibody that binds the extracellular domain of the epidermal growth factor receptor (EGFR). It is a 152kD 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.
Sample Type
Serum and plasma
Intended Use
The Cetuximab PK BioAssay™ ELISA kit is designed to measure cetuximab in serum or plasma with high specificity and sensitivity. This sandwich ELISA design employs a Cetuximab specific monoclonal antibody for capture and a specific monoclonal antibody for detection.
Recovery
1000ng/ml of Cetuximab was spiked in 10 lots of human serum. Recovery ranges are from 91-113% with an average recovery of 106%.
Precision:t|Intra-assay CV: <10% |Inter-assay CV: <10%.
Test Principle
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.
Kit Components
Coated microtiter plate, 1x96 wells Calibrator diluent, 1x1.8mlCalibrator (0.2mg/ml), 1x12ulWash buffer, 10X, 1x25mlAssay buffer 1x50mlDetection reagent, 1000X, 1x17ulTMB, 1x12mlTMB stop solution, 1x12ml
Storage and Stability
Store kit components at -20°C unless specified otherwise. Unopened kit is stable for 6 months after receipt. For maximum recovery of product, centrifuge small tubes on pulse setting prior to opening.
