Highly specific and sensitive reagents for antibody drug development assays.
Technical articles featuring applications of anti-idiotypic antibodies.
Ready-made recombinant, anti-idiotypic monoclonal antibodies, highly specific for the chimeric antibody drug cetuximab (Erbitux).
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.
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.
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
Table 1. Antibodies Specific to Cetuximab
|Catalog #||Clone||Antibody Specificity||Binding Type||Format||Affinity KD, nM||Assay Development Recommendations|
PK bridging ELISA
capture antibody with HCA221 or HCA228
PK bridging ELISA detection antibody with HCA220
|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|
|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
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
Fig. 1. PK bridging ELISA measuring free cetuximab HCA220 & HCA221P.
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.
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)
Fig. 2. ADA bridging ELISA using antibody HCA221, HCA222 or HCA223.
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
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.
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.
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.
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