Cetuximab (Erbitux) Pharmacokinetic ELISA

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.

Product Specifications

CAS Number

7732-18-5

Gene ID

1956

Reactivity

Human, mouse, rat

Reaction Volume

15 µL

Target Antigen

Cetuximab (Erbitux)

Detection Method

Peroxidase / OD450

Assay Type

ELISA

Assay Principle

Quantification of Cetuximab in biological matrices

Assay Protocol

The Cetuximab ELISA kit is designed to measure free Cetuximab with high specificity and sensitivity . This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Cetuximab 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Cetuximab present in biological matrices is bound by the immobilized capture antibody. After washing away any unbound substances, enzyme linked detection antibody is added to the wells. 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 and the concentration is calculated from the standard series.

Assay Performance Time

2.5 hours

Sample Type

Serum Plasma

Sample Volume

15ul

Detection Range

50ng/ml - 1.56ng/ml

Precision

<10%, <10%

Sensitivity

Cetuximab

Limit Of Detection

1.5ng/ml

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

Shipping Conditions

Blue Ice

Storage Conditions

-20C, 1 year

Shelf Life

12 months

Specificity

Cetuximab

Method

Direct sandwich ELISA

Preservative

None

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.

Operating Procedure

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.

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.