Optimal 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®).
The Type 1 anti-cetuximab antibodies inhibit the binding of the drug cetuximab to its target, EGF Receptor (EGFR), and therefore detect free drug. These antibodies are ideal for use in direct ELISA, pharmacokinetic (PK) bridging assays and to develop and calibrate immune response (IR) assays to measure the anti-drug antibody (ADA) response in patient sera.
Product codes: HCA220, HCA221, HCA221P, HCA222, HCA223
The Type 2 anti-cetuximab antibody does not inhibit the binding of cetuximab to EGFR and can therefore detect total drug – free, partially bound and fully bound. This fully human antibody can be used to measure the cetuximab levels in patient sera.
Product code: HCA228, HCA228P
The recombinant anti-cetuximab antibodies are generated using the HuCAL® antibody library and a modified and improved form of phage display. Antibody generation using HuCAL technology results in highly specific and sensitive antibodies that are ideal for PK and PD assays. Antibodies are fully human and are therefore appropriate as controls or calibrators for ADA assays, replacing the need for polyclonal sera. 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 antibodies and their binding types and properties
|Product Code||Clone||Antibody Specificity||Binding Type||Format||Affinity KD, nM||Assay Development Recommendations|
PK bridging ELISA
Capture antibody with HCA221 or HCA228
|HCA221||AbD19830_IgG1||Cetuximab||Type 1||Human IgG1||1.7*||
PK bridging ELISA with HCA220
|1.7*||PK bridging ELISA with HCA220|
|HCA222||AbD19834_IgG1||Cetuximab||Type 1||Human IgG1||0.5*||
|HCA223||AbD19815_IgG1||Cetuximab||Type 1||Human IgG1||10.5*||
|HCA228||AbD19376_IgG1||Infliximab2||Type 2||Human IgG1||39*||
PK bridging ELISA with HCA220
PK bridging ELISA with HCA220
Table 1 Antibody Specifications
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.
*Affinity measured in the monovalent Fab format
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
Figure 1: PK assay, bridging format, using antibodies HCA220 & HCA221
Figure 1: Anti-cetuximab antibody, clone AbD19834 (HCA220) was coated on a microtiter plate at 1.0 µg/ml and left over night. 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) diluted to 2 µg/ml in HISPEC buffer (BUF049), plus QuantaBlu™ fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
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.
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)
Figure 2: ADA assay bridging format, using antibodies HCA221, HCA222 & HCA223
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 buffer (BUF042), followed by QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
Figure 3: Inhibition of cetuximab binding to EGFR
Figure 3: EGFR was coated on a microtiter plate at 1.0 µg/ml, followed by a pre-incubated mixture of the anti-cetuximab antibodies HCA220, HCA221 and HCA223 (inhibitory), and HCA228 (non-inhibitory) titrated into a constant amount of cetuximab (0.3 µg/ml). HCA221, HCA223 and HCA228 used in this assay are in monovalent Fab-FH format. Free cetuximab still capable of binding to the EGFR was detected using HRP conjugated anti-human Fab antibody. Data are shown as the mean of three measurements.
Figure 4: Demonstration of the specificity of anti-cetuximab antibody HCA222
Figure 4: Specificity ELISA for type 1 anti-cetuximab antibody HCA222 using various antigens as coating reagents. Bound HRP conjugated anti cetuximab HCA222* was directly detected using QuantaBlu as substrate.
Figure 5: Specificity of anti-cetuximab antibody HCA228
Figure 5: Cetuximab and other antigens were immobilized on a microtiter plate. After washing and blocking with 5% BSA, anti-cetuximab HCA228 (Fab-FH format) was titrated in PBST plus 10% human serum. Detection was performed using HRP conjugated anti-Penta Histidine tag 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.
* Antibodies were conjugated to HRP using a LYNX Rapid Conjugation Kit® (product codes LNK001P - LNK006P)
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.
ERBITUX® is a registered trademark of ImClone LLC. HuCAL® is a registered trademark of MorphoSys AG. QuantaBlu™ is a trademark of Thermo Fisher Scientific. LYNX Rapid Conjugation Kit® is a registered trademark of Bio-Rad AbD Serotec Ltd.