AffinityImmuno · ELISA Kit
Cetuximab (Erbitux®) Pharmacokinetic ELISA Kit
Cat.No
EL-1611-031
1X96 wells
- Reactivity
- Human, mouse, rat
- Detection range
- 50ng/ml - 1.56ng/ml
- Sample type
- Serum Plasma
- Assay time
- 2.5 hours
- Antigen
- Cetuximab (Erbitux)
- Detection minimum
- 1.5ng/ml
Product Overview
This Cetuximab (Erbitux®) kit includes a chimeric IgG1 monoclonal antibody that binds the extra-cellular domain of the epidermal growth factor receptor.
INTRODUCTION
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.
Specifications
- Gene ID
- 1956
- Reactivity
- Human, mouse, rat
- Detection range
- 50ng/ml - 1.56ng/ml
- Sample type
- Serum Plasma
- Sample volume
- 15ul
- Assay time
- 2.5 hours
- Assay precision
- <10%, <10%
- Storage
- -20C, 1 year
- Size
- 1X96 wells
- Antigen
- Cetuximab (Erbitux)
- Detection minimum
- 1.5ng/ml
- Method
- Direct sandwich ELISA
- Detection method
- Peroxidase / OD450
- Application
- Quantification of Cetuximab in biological matrices
- Plate format
- Strip
- Specificity
- Cetuximab
- Preservative
- None
Kit Components
- Coated microtiter plate, 96 wells
- Calibrator diluent. - 1.8ml
- Calibrator 12ul
- 10X wash buffer - 25ml
- Assay buffer - 50ml
- 1000X detection reagent - 17ul
- TMB - 12ml
- TMB stop solution - 12ml
- Plate sealers - 3
Assay Information
Reagent preparation
Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation Dilute wash buffer concentrate with deionized water 1/10 before use (for example add 20mL concentrate to 180mL deionized water). Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 11μl concentrate to 11ml of assay buffer). Mix well. The following is an example calibrator curve.
Results calculation
1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. Alternatively a log-log curve fit may be used. 2. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 3. Any sample undiluted or diluted still reading greater than the highest standard should be diluted appropriately with assay buffer and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.
Sample collection
This kit is compatible with EDTA-plasma, heparinplasma and serum samples. Samples can be stored at or below -20°C for up to 1 year.
Sample preparation
Dilute calibrators and test samples 1/100 with assay buffer (for example add 5µL of prepared calibrator or sample to 495µL of assay buffer). Mix well. Do not store diluted samples.