Anti-Cetuximab Antibodies

Ideal for bioanalytical assays to measure the levels of cetuximab and biosimilar products in preclinical research and clinical trials

Ready-made recombinant, anti-idiotypic monoclonal antibodies, highly specific for the chimeric antibody drug cetuximab (Erbitux).

Anti-Cetuximab Type 1 Antibodies

Anti-Cetuximab Inhibitory Antibodies

These inhibitory (Type 1) antibodies are specific for cetuximab and prevent it binding to its target, epidermal growth factor receptor (EGF R), and can therefore be used to detect free drug. They are ideal as capture and detection reagents in pharmacokinetic (PK) bridging assays and as a surrogate positive control for an anti-drug antibody (ADA) assay.

Catalog Numbers: HCA220, HCA221, HCA221P, HCA222, HCA223

Anti-Cetuximab Type 2 Antibodies Anti-Cetuximab Non-Inhibitory Antibody

The non-inhibitory (Type 2) anti-cetuximab antibody does not inhibit the binding of cetuximab to EGF R and can therefore detect total drug – free, partially bound and fully bound. This antibody can be used in a PK assay to measure total drug levels in patient sera.

Catalog Numbers: HCA228, HCA228P

Our recombinant, monoclonal antibodies are generated using the Human Combinatorial Antibody Library (HuCAL®) and CysDisplay®, a proprietary method of phage display, with guided selection methods to obtain highly targeted reagents. The result is highly specific and high affinity antibodies, ideal for development of PK assays. These fully human antibodies are also suitable as controls or calibrators for ADA assays. The in vitro production of recombinant antibodies means that there is a consistent, secure supply throughout preclinical development and clinical trials, avoiding the need to develop new reagents.

More information about anti-idiotypic antibody binding types 1, 2 and 3 and their properties

Anti-biotherapeutic antibody quality control and characterization


Table 1. Antibodies Specific to Cetuximab

Catalog # Clone Antibody Specificity Binding Type Format Affinity KD, nM Assay Development Recommendations
HCA220 AbD19834 Cetuximab Inhibitory Fab-FH1 0.5 PK bridging ELISA
capture antibody with HCA221 or HCA228
HCA221 AbD19830_IgG1 Cetuximab Inhibitory Human IgG1 1.73 PK bridging ELISA detection antibody with HCA220
ADA assay
HCA221P AbD19830_IgG1 Cetuximab Inhibitory Human IgG1
HRP labeled
1.73 PK bridging ELISA detection antibody with HCA220
HCA222 AbD19834_IgG1 Cetuximab Inhibitory Human IgG1 0.53 ADA assay
HCA223 AbD19815_IgG1 Cetuximab Inhibitory Human IgG1 10.53 ADA assay
HCA228 AbD19376_IgG1 Infliximab2 Non-Inhibitory Human IgG1 393 PK bridging ELISA detection antibody with HCA220
HCA228P AbD19376_IgG1 Infliximab2 Non-Inhibitory Human IgG1
HRP labeled
393 PK bridging ELISA detection antibody with HCA220

1 F=DYKDDDDK-tag H=His-6-tag;
2 HCA228 binds to an epitope outside the infliximab binding site. This epitope is shared with the chimeric antibody cetuximab.
HCA228 binds equally well to the chimeric antibodies cetuximab and infliximab.
3 Affinity measured in the monovalent Fab format

Related Products

Cetuximab drug isotype control Recombinant Human IgG1 Kappa (HCA192)
Cetuximab biosimilar for research use Human Anti-EGFR Antibody (MCA6102)


Pharmacokinetic Assay - Bridging ELISA

Pharmacokinetic (PK) Assay - Bridging ELISA

 

Schematic image of PK Bridging ELISA. Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold), anti-idiotypic detection antibody, Ig format (blue), labeled with HRP

PK assay, bridging format, using antibodies HCA220 & HCA221

Fig. 1. PK bridging ELISA measuring free cetuximab HCA220 & HCA221P.

Measurement of free or total drug with anti-cetuximab antibodies

In figure 1, Anti-Cetuximab Antibody clone AbD19834 (HCA220) was coated on a microtiter plate at 1.0 µg/ml and left overnight. Washing and blocking was performed with 5% BSA in PBST. Cetuximab titrated into 10% human serum was added. Detection was performed using HRP conjugated Anti-Cetuximab Antibody clone AbD19830_hIgG1 (HCA221P) at 2 µg/ml in HISPEC Assay Diluent (BUF049), plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: PK bridging ELISA measuring free cetuximab HCA220 and HCA221P

Protocol: PK bridging ELISA measuring total cetuximab HCA220 and HCA228P


Anti-Drug Antibody Assay - Bridging ELISA

Three anti-cetuximab antibodies in IgG1 format are available, each with a different affinity for the target, allowing the ADA assay to be tailored to the specific needs of the developer.

 

Anti-Drug Antibody (ADA) Assay - Bridging ELISA

Schematic image of ADA bridging assay. Monoclonal antibody drug as capture antibody and detection antibody labeled with HRP (gold), fully human anti-idiotypic antibody, Ig format (blue)

ADA assay, bridging format, using antibodies HCA221, HCA222 & HCA223

Fig. 2.  ADA bridging ELISA using antibody HCA221, HCA222 or HCA223.

Immunogenicity assay using human anti-cetuximab antibody as calibration standard

In figure 2, cetuximab was coated at 1 µg/ml on a microtiter plate and left overnight. After washing and blocking with 5% BSA in PBST, the Anti-Cetuximab Antibody HCA221, HCA222 or HCA223 spiked in 10% human serum was added in the given concentrations. Detection was performed by adding HRP conjugated cetuximab at 2 µg/ml in HISPEC Assay Diluent (BUF042), followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. HRP conjugation of cetuximab was performed using a LYNX Rapid HRP Antibody Conjugation Kit.

Protocol: ADA bridging ELISA protocol cetuximab


Inhibition ELISA

 Inhibition of cetuximab binding to EGFR

Fig. 3. Inhibition of cetuximab binding to EGF R.

In figure 3, EGF R was coated on a microtiter plate at 1.0 µg/ml, followed by a pre-incubated mixture of the Anti-Cetuximab Antibodies HCA220, HCA221 or HCA223 (inhibitory), or HCA228 (non-inhibitory), titrated into a constant amount of cetuximab (0.3 µg/ml). HCA221, HCA223 and HCA228 used in this assay were in monovalent Fab-FH format. Free cetuximab still capable of binding to the EGF R coated plate was detected using HRP conjugated anti-human Fab antibody. Data are shown as the mean of three measurements.


Demonstration of Antibody Specificity

Demonstration of the specificity of anti-cetuximab antibody HCA222

Fig. 4. Specificity of Type 1 Anti-Cetuximab Antibody HCA222.

In figure 4, a specificity ELISA is shown for type 1 Anti-Cetuximab Antibody HCA222 using various antigens as coating reagents. Bound HRP conjugated antibody was directly detected using QuantaBlu Fluorogenic Peroxidase Substrate.


Specificity Titration ELISA

Specificity of anti-cetuximab antibody HCA228
Fig. 5. Specificity of Type 2 Anti-Cetuximab Antibody HCA228.

In figure 5, cetuximab and other antigens were immobilized on a microtiter plate. After washing and blocking with 5% BSA, Anti-Cetuximab Antibody HCA228 (Fab-FH format) was added, titrated in PBST plus 10% human serum. Detection was performed using Mouse Anti-Penta Histidine Tag:HRP Antibody (MCA5995P) and QuantaBlu Fluorogenic Peroxidase Substrate.

The Type 2 antibody, HCA228, binds to an epitope outside the binding site of the antibody drug. The immunogen for HCA228 was infliximab, and this particular epitope is shared with other chimeric antibodies including cetuximab. This results in HCA228 recognizing both infliximab and cetuximab. HCA228 does not cross-react with the chimeric antibody rituximab.


 

Licensed Use: For in vitro research purposes and for commercial applications for the provision of in vitro testing services to support preclinical and clinical drug development. Any re-sale in any form or any other commercial application needs a written agreement with Bio-Rad.