Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Kit

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SKU:
HUMB00034
€899

Description

Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Kit

Enzyme-linked immunosorbent assay for the qualitative determination of antibodies to Cetuximab in serum and plasma. Cetuximab (Erbitux®) was associated to the development of anti-cetuximab antibodies, with some reported to be neutralizing in patients. The ELISA Genie Antibody to Cetuximab ELISA Kit can be efficiently used for monitoring cetuximab anti-drug antibodies (ADA) and is for research use only.

Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Kit test principle

The ELISA Genie Antibody to cetuximab (Erbitux®) ELISA is a sandwich assay for the determination of antibodies against cetuximab in serum and plasma samples. During the first incubation period, antibodies to cetuximab (ATC) in patient serum/ plasma samples are captured by the drug cetuximab (Erbitux®) coated on the wall of the microtiter wells. After washing away the unbound components from samples, a peroxidase-labelled specific conjugate is added to each well and then incubated. After a second washing step, the bound enzymatic activity is detected by addition of tetramethylbenzidine (TMB) chromogen-substrate. Finally, the reaction is terminated with an acidic stop solution. The intensity of the reaction colour is directly proportional to the concentration of ATC in sample.

Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Product Information

Information Description
Application
Free drug
Required Volume (μl)
10
Total Time (min)
140
Sample Type
Serum, Plasma
Number of Assays
96
Detection Limit (ng/mL)
plus/minus
Spike Recovery (%)
-
Shelf Life (year)
1

Alternative Names

Anti-Epidermal Growth Factor Receptor (EGFR) mAb

Erbitux

Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA - Key Information

Cetuximab (Erbitux®) mode of action  

Cetuximab (IMC-C225, Erbitux™) is a human/murine chimeric monoclonal antibody of the immunoglobulin G1 (IgG1) and FDA-approved epidermal growth factor receptor (EGFR) inhibitor. It is a 152 kDa protein composed of four polypeptide chain. There are 32 cysteine residues forming accordingly 16 potential disulfide bonds.

Cetuximab (ICM-225, Erbitux™) works by binding to the extracellular domain III of EGFR. This binding partially blocks the EGFR ligand-binding domain and sterically hinders the correct extended conformation of the dimerization arm on domain II. Cetuximab therefore prevents both ligand binding and the proper exposure of the EGFR dimerization domain, which hinders dimerization with other HER family members.

Cetuximab (Erbitux®) uses

Preclinical studies have shown that cetuximab enhances the anti-tumour effects of chemotherapy (e.g. that of irinotecan in colorectal cancer) as well as radiotherapy (e.g. in squamous cell carcinoma of the head and neck) by inhibiting cell proliferation, angiogenesis and metastasis and by promoting apoptosis is used for the treatment of metastatic colorectal cancer, metastatic non-small cell lung cancer and head and neck cancer.

Cetuximab blocks growth factor-induced activation of the downstream mitogen-activated protein kinase, inhibiting cell proliferation. It has also been shown that cetuximab increases the receptor internalization which is another mechanism to silence the receptor. Cetuximab arrests cell cycle at G1 gap phase by upregulating antiproliferative p27 kip1 , which functions via complex formation with Cdk2, and downregulating proliferating cell nuclear antigen (PCNA).

Cetuximab decreases angiogenic factors, inhibits tumor-cell invasion and metastasis via downregulation of matrix metalloproteinases (MMPs) and VEGF, and promotes apoptosis by upregulating apoptotic protein, Bax, with the help of other chemotherapeutic agents. Cetuximab also displays synergistic effect with other agents and/or radiotherapy. Binding of antigen-binding fragment (Fab) of Cetuximab, which displays higher affinity comparing to ligands of EGFR, takes place via domain III of extracellular EGFR, preventing the receptor from conformational change to be dimerized and blocking EGFR signalling through inhibition of EGF and TGF-alpha-stimulated phosphorylation of the receptor.

Cetuximab (Erbitux®) and Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) Epidermal growth factor receptor (EGFR; HER1; ErbB1) is a transmembrane tyrosine kinase encoded by c-erb-B proto-oncogene, expressed in normal and malignant cells and stimulated by epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) binding extracellular domain of the receptor, leading receptor to dimerize and activating intracellular kinase domain on each receptor, bringing about phosphorylation of tyrosine residues on each member of the receptor pair. Next, signalling complexes form in cytoplasm to activate gene transcription responding for such as cell proliferation. Termination of signalling occurs through internalization of receptor-ligand complex. Activation of EGFR results in perturbation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase, and AKT pathways triggering tumorigenic processes, such as increased proliferation, angiogenesis and metastasis, and prevents apoptosis.

Breast, lung, colon, prostate, kidney, bladder, head and neck, and ovary cancers have been associated to EGFR overexpression which causes early disease progression, poor survival, and resistance to chemotherapy in many epithelial malignancies. Epidermal growth factor receptor/human epidermal growth factor receptor 1 (EGFR/HER1) and its ligand, transforming growth factor alpha (TGF-alpha) were showed to involve in hepatocarcinogenesis. EGFR is overexpressed in hepatocellular carcinoma (HCC). To overcome the uncontrollable effect of EGFR triggering cancer development, monoclonal antibodies, such as Cetuximab, have been shown to be used as blockers in vitro and in vivo.

Cetuximab (Erbitux®) immunogenicity  

As with any biologic therapeutic, immunogenicity, in the form of anti-cetuximab antibodies can occur. The demonstration of anti-cetuximab antibodies during treatment with cetuximab (Erbitux®) is a major concern. The ELISA Genie Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Kit can be efficiently used for monitoring anti-cetuximab antibodies in biological samples and is for research use only.

Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Kit Contents

Size Kit Contents

1 x 12 x 8

Microtiter Plate

Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant

1 x 0.25 mL

Reactive Control
Ready-to-use. Contains cetuximab -reactive reagent, human serum, stabilizers
and <0.1% NaN3

1 x 0.5 mL

Negative Control
Ready-to-use. Contains human serum, stabilizers and <0.1% NaN3

1 x 50 mL

Assay Buffer
Blue coloured. Ready to use. Contains proteins and <0.1% NaN3.

1 x 12 mL

Peroxidase Conjugate

Red coloured. Ready to use. Contains HRP-probe, stabilizer and preservatives.

1 x 12 mL

TMB Substrate Solution
Ready to use. Contains TMB

1 x 12 mL

TMB Stop Solution
Ready to use. 1N HCl

1 x 50 mL

Wash Buffer concentrate (20x)
Contains Buffer with Tween 20.

2 x 1

Adhesive Foil
For covering of Microtiter Plate during incubation.


Anti-Cetuximab (Erbitux®) ADA Qualitative ELISA Protocol

Steps Protocol

1

Pipette 100µl of Assay Buffer non-exceptionally into each of the wells to be used.

2

QUALITATIVE ELISA TEST FORMAT
Pipette 10 µL of ready-to use Negative Control, Reactive Control, and Samples into the respective
wells of microtiter plate.
Wells
A1: Negative Control
B1: Negative Control
C1: Reactive Control
D1 and on: Sample (Serum/Plasma)

3

Cover the plate with adhesive film. Briefly mix contents by gently shaking the plate. Incubate 60 min
at room temperature (18-25°C).

4

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300µL of diluted. Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

5

Pipette 100 µL of ready-to use Peroxidase Conjugate into each well.

6

Cover the plate with adhesive foil. Incubate 60 min at room temperature (18- 25°C).

7

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µL of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

8

Pipette 100 µL of TMB Substrate Solution into each well.

9

Incubate 20 min (without adhesive foil) at room temperature (18-25°C) in the dark

10

Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Colour changes from blue to yellow.

11

Measure optical density with a photometer at 450/650 nm within 30 min after pipetting of the Stop Solution.

Trademarks

Erbitux® CETUXIMAB is a trademark of ImClone LLC

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