Antibodies for bioanalytical method development to measure trastuzumab and biosimilar products
Ready-made recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized antibody drug trastuzumab (Herceptin)
The Type 1 anti-trastuzumab antibodies inhibit the binding of the drug Herceptin to its target, the extracellular domain of the human epidermal growth factor receptor 2 (HER2, also known as ErbB2), and therefore detect free drug. These antibodies are ideal for use in direct and indirect 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: HCA166, HCA167, HCA168, HCA169, HCA176(P), HCA177(P), HCA270 (IgG4 isotype)
The Type 3 antibody specifically recognizes the drug-target complex. It detects trastuzumab only when it is bound to HER2. This antibody does not bind to free trastuzumab or unbound HER2. It can be used to set up a PK antigen capture assay that avoids the bridging format. In addition, it allows detection of trastuzumab bound to its target in serum as opposed to free trastuzumab.
Product code: HCA263
These recombinant monoclonal antibodies are generated using the HuCAL® phage display antibody library, which results in highly specific and sensitive reagents, ideal for PK assays. Antibodies are fully human, so in full immunoglobulin format they can be used as reference standards 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 antibodies and their binding types and properties
|Product Code||Clone||Antibody Specificity||Binding Type||Format||Affinity KD, nM||Assay Development Recommendations|
PK bridging ELISA with HCA168
PK bridging ELISA
PK bridging ELISA with HCA166
PK bridging ELISA with HCA176P
|HCA176||AbD16712_hIgG1||Trastuzumab||Type 1||Human IgG1||0.43||
PK bridging ELISA with HCA169
PK bridging ELISA with HCA169
|HCA177||AbD18018_hIgG1||Trastuzumab||Type 1||Human IgG1||0.023||
PK bridging ELISA
PK bridging ELISA
PK bridging ELISA with anti-IgG4
|HCA263||AbD25279||Trastuzumab/HER2||Type 3||Fab-A-FH4||313||PK ELISA antigen capture format|
Table 1 Antibody Specifications
1 Monovalent Fab antibody, V5- and StrepX-StrepX- tags
2 Monovalent Fab antibody DYKDDDDK- and His-6-tags
3 Affinities measured in the monovalent Fab format
4 Bivalent mini antibody, bivalent by dimerization of the bacterial alkaline phosphatase fusion protein, DYKDDDDK- and His-6-tags
5 IgG4Pro isotype has a point mutation to prevent Fd half molecule exchange
Schematic image of PK Bridging ELISA. Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold), anti-idiotypic detection antibody labeled with HRP
Figure 1: PK assay, bridging format, using antibodies HCA168 (capture) and HCA166 (detection)
Figure 1: Anti-trastuzumab antibody, clone AbD18018 (HCA168) was coated at 1 μg/ml on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, trastuzumab in the given concentrations was added in PBST plus 10% human serum. Detection was performed by HRP conjugated anti-trastuzumab antibody, clone AbD16712 (HCA166)*, at a concentration of 4 µg/ml in HISPEC assay diluent, plus QuantaBlu fluorogenic peroxidase substrate. Data are mean values of three experiments. HCA166 and HCA168 can be used interchangeably as cpature and detection antibodies in this bridging ELISA with a similar result.
Schematic image of PK Bridging ELISA (IgG4). Anti-idiotypic capture antibody, Fab format (purple), monoclonal antibody drug (gold), detection with anti-idiotypic antibody IgG4 isotype format (blue), and anti-human IgG4 labeled with HRP (gray).
Figure 2: PK assay, bridging format; using antibodies HCA169 (capture), and HCA270 (detection) with HRP conjugated anti-hIgG4, MCA2098P
Anti-trastuzumab antibody clone AbD18018 has been converted to human IgG4 isotype to offer assay developers increased assay design flexibility. When used as the primary detection antibody, a secondary anti-human IgG4 antibody is needed, labeled according to the requirements of the assay platform.
Figure 2: Anti-trastuzumab antibody, clone AbD18141 (HCA169) was coated at 1 μg/ml on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, 10% human serum spiked with increasing concentrations of trastuzumab was added. Detection was performed by (a) HRP conjugated anti-trastuzumab antibody, clone AbD18018_hIgG4Pro* (HCA270) at a concentration of 2 µg/ml in HISPEC assay diluent (black), or (b) clone AbD18018_hIgG4Pro (2 µg/ml) followed by HRP conjugated mouse anti-human IgG4 antibody (MCA2098P) at a 1:10,000 dilution (green); plus QuantaBlu fluorogenic peroxidase substrate.
Schematic image of PK antigen capture format ELISA. Drug target (red), monoclonal antibody drug (gold), drug target complex detection antibody, Fab monovalent format (purple), labeled with HRP
Figure 3: Trastuzumab PK ELISA antigen capture format using antibody HCA263
Figure 3: Human ErbB2 was coated over night on a microtiter plate at 5 µg/ml. After washing and blocking with 5% BSA in PBST, 10% human serum was added, spiked with increasing concentrations of trastuzumab. Detection was performed using the bivalent mini antibody, human anti-trastuzumab/HER2 complex-specific antibody, clone AbD25279 (HCA263) at a concentration of 2 μg/ml, followed by HRP labeled mouse anti-Penta Histidine tag antibody (MCA5995P) in HISPEC assay diluent (BUF049A) and QuantaBlu fluorogenic peroxidase substrate. Data is presented as the mean of three measurements. The antibody was conjugated to HRP using a LYNX Rapid Conjugation Kit® (LNK001P-LNK006P).
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 4: Immunogenicity assay, bridging format, using antibody HCA177
Figure 4: Trastuzumab was coated at 0.5 µg/ml on a microtiter plate and left overnight. After washing and blocking with 5% BSA in PBST, anti-trastuzumab antibody, clone AbD18018_hIgG1 (HCA177), titrated in 10% human serum in the given concentrations, was added. Detection was performed by adding HRP conjugated trastuzumab, 2.0 µg/ml in HISPEC assay diluent (BUF049), plus QuantaBlu fluorogenic peroxidase substrate.
Figure 5: Demonstration of the specificity of antibody HCA166 to trastuzumab
Figure 5: Antigens were coated at 5.0 µg/ml on a microtiter plate and left overnight. After washing and blocking with 5% BSA, anti-trastuzumab antibody, clone AbD16712 (HCA166), was added at a concentration of 2.0 µg/ml. Detection was performed by adding anti-Strep-tag antibody, HRP conjugated (MCA2489P) in HISPEC assay diluent plus QuantaBlu fluorogenic peroxidase substrate
* Antibody 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.