Anti-Trastuzumab Antibodies

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®)

Type 1 anti-idiotypic antibody

Anti-Trastuzumab Type 1 Antibodies

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)

Type 3 anti-drug-target complex specific antibody

Anti-Trastuzumab Drug/Target Complex Type 3 Antibody

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

Anti-biotherapeutic antibody quality control and characterization


Antibodies Specific to Trastuzumab

Product Code Clone Antibody Specificity Binding Type Format Affinity KD, nM Assay Development Recommendations
HCA166 AbD16712 Trastuzumab Type 1 Fab-V5Sx21 0.4 Indirect ELISA
PK bridging ELISA with HCA168
HCA167 AbD16714 Trastuzumab Type 1 Fab-V5Sx21 2.5 Indirect ELISA
PK bridging ELISA
HCA168 AbD18018 Trastuzumab Type 1 Fab-FH2 0.02 Indirect ELISA
PK bridging ELISA with HCA166
HCA169 AbD18141 Trastuzumab Type 1 Fab-FH2 0.07 Indirect ELISA
PK bridging ELISA with HCA176P
HCA176 AbD16712_hIgG1 Trastuzumab Type 1 Human IgG1 0.43 Indirect ELISA
PK bridging ELISA with HCA169
ADA assay
HCA176P AbD16712_hIgG1 Trastuzumab Type 1 Human IgG1
HRP labeled
0.43 Direct ELISA
PK bridging ELISA with HCA169
HCA177 AbD18018_hIgG1 Trastuzumab Type 1 Human IgG1 0.023 Indirect ELISA
PK bridging ELISA
ADA assay
HCA177P AbD18018_hIgG1 Trastuzumab Type 1 Human IgG1
HRP labeled
0.023 Direct ELISA
PK bridging ELISA
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
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


Pharmacokinetic Assay – Bridging ELISA

Schematic image of PK Bridging ELISA.

 

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

PK assay, bridging format, using antibodies HCA168 (capture) and HCA166 (detection)

Figure 1: PK assay, bridging format, using antibodies HCA168 (capture) and HCA166 (detection)

Human anti-trastuzumab antibodies, product codes HCA166 and HCA168

Figure 1:  Anti-trastuzumab antibody 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.

Protocol: PK bridging ELISA for use with anti-trastuzumab antibodies HCA166, HCA168

Protocol: PK bridging ELISA for use with anti-trastuzumab antibodies HCA169, HCA176P

* HCA166 was conjugated to HRP using a LYNX Rapid Conjugation Kit® (product codes LNK001P - LNK006P)


Pharmacokinetic Assay – Antigen Capture Format

Schematic image of anti-drug/target complex specific antibody in PK ELISA antigen capture format

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

Adalimumab PK ELISA antigen capture format using antibody HCA231

Figure 2: Trastuzumab PK ELISA antigen capture format using antibody HCA263

Human anti-trastuzumab antibody, product code HCA263

Figure 2: 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).

Protocol: PK antigen capture ELISA protocol trastuzumab


Anti-Drug Antibody Assay – Bridging ELISA

Schematic image of ADA bridging assay

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)

Immunogenicity assay, bridging format, using antibody HCA177

Figure 3: Immunogenicity assay, bridging format, using antibody HCA177

 

Human anti-trastuzumab antibody, product code HCA177

Figure 3: 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.

Protocol: ADA bridging ELISA for use with anti-trastuzumab antibody


Demonstration of Antibody Specificity

 

Demonstration of the specificity of antibody HCA166 to trastuzumab

Figure 4: Demonstration of the specificity of antibody HCA166 to trastuzumab

Human anti-trastuzumab antibody, product code HCA166

Figure 4: 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


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.

Herceptin® is a registered trademark of Genentech, Inc. HuCAL® is a registered trademark of Morphosys AG.  QuantaBlu™ is a trademark of Thermo Fisher Scientific Inc.  LYNX Rapid Conjugation Kit® is a registered trademark of Bio-Rad AbD Serotec Ltd.