Cetuximab ELISA Kit

Catalog No. ABIN4886391

Product Details Cetuximab ELISA Kit

Target: Cetuximab

Reactivity: Human, Mouse, Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.56-50 ng/mL

Minimum Detection Limit: 1.56 ng/mL

Application: ELISA

Purpose: Quantification of Cetuximab in biological matrices

Sample Type: Plasma, Serum

Analytical Method: Quantitative

Specificity: Cetuximab (Erbitux)

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

Material not included: Precision pipettes calibrated to deliver 5-1000μL
Multi-channel pipette calibrated to deliver 50-200μL
Plate shaker
Disposable tips
Vortex-Mixer
Distilled or de-ionized water
Microplate reader capable of reading 450nm with background subtrac

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Sample Volume: 15 μL

Assay Time: 2.5 h

Plate: Pre-coated

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.

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 20 mL concentrate to 180 mL deionized water). Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 11 μL concentrate to 11 mL of assay buffer). Mix well. 3. Preparation of Calibrators: Prepare calibrators with concentrations ranging from 5,000 ng/mL to 156 ng/mL. The following is an example calibrator curve.

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.

Assay 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.

Calculation of Results:

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 calibrator diluent and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.

Assay Precision: Precision: The precision was determined by analyzing samples prepared at 500 ng/mL in 6 replicates on 6 different occasions. Intra-assay coefficient of variation (CV) < 10%. Inter-assay CV < 10%.

Recovery: 1000 ng/mL of Cetuximab was spiked in 10 lots of human serum. Recovery ranges are from 91-113% with an average recovery of 106%.

Restrictions: For Research Use only

Handling

Preservative: Without preservative

Precaution of Use: Read manual completely before beginning

Storage: -20 °C

Storage Comment: Store kit components at -20°C unless specified otherwise. DO NOT USE past kit expiration date. Some vials contain a small amount of reagents. Spin tubes on pulse setting prior to opening.

Expiry Date: 12 months

References for Cetuximab ELISA Kit (ABIN4886391)

van Helden, Elias, Gerritse, van Es, Boon, Huisman, van Grieken, Dekker, van Dongen, Vugts, Boellaard, van Herpen, de Vries, Oyen, Brouwers, Verheul, Hoekstra, Menke-van der Houven van Oordt: "[89Zr]Zr-cetuximab PET/CT as biomarker for cetuximab monotherapy in patients with RAS wild-type advanced colorectal cancer." in: European journal of nuclear medicine and molecular imaging, Vol. 47, Issue 4, pp. 849-859, (2021) (PubMed).

Target Details for Cetuximab

Target: Cetuximab

Abstract: Cetuximab Products

Target Type: Antibody

Background: 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.

Gene ID: 1956