Anti-Bevacizumab Antibodies

Antibodies for bioanalytical assays to measure bevacizumab and biosimilar products

Ready-made recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized antibody drug bevacizumab (Avastin®).

Anti-Bevacizumab Antibodies

Anti-Bevacizumab Type 1 Antibodies

The type 1 anti-bevacizumab antibodies 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: HCA182, HCA184, HCA184P, HCA185

These recombinant, monoclonal, anti-idiotypic antibodies are generated using the HuCAL® antibody library and a proprietary method of phage display, which results in highly specific and sensitive reagents, ideal for PK and PD assays. Because these antibodies are fully human, they are also perfect as reference standards for ADA assays when in full immunoglobulin format. The in vitro production of recombinant antibodies results in a consistent, secure supply of these critical reagents throughout preclinical development and clinical trials.

More information about anti-idiotypic antibodies and their binding types and properties

Anti-biotherapeutic antibody quality control and characterization


Antibodies Specific to Bevacizumab

Product Code Clone Antibody Specificity Binding Type Format Affinity KD, nM Assay Development Recommendations
HCA182 AbD17976 Bevacizumab Type 1 Fab-V5Sx21 0.4 Indirect ELISA
PK bridging ELISA with HCA184P

HCA184

AbD16748_hIgG1

Bevacizumab

Type 1

Human IgG1

2.12

Indirect ELISA
PK bridging ELISA with HCA182

HCA184P

AbD16748_hIgG1 Bevacizumab Type 1 Human IgG1
HRP labeled
  Direct ELISA
PK bridging ELISA with HCA182

HCA185

AbD17976_hIgG1

Bevacizumab

Type 1

Human IgG1

0.42

Indirect ELISA
ADA assay

Table 1  Antibody Specifications
1 Monovalent Fab antibody (V5-tag and StrepX-StrepX-tag)
2 Affinity measured in the monovalent Fab form


Pharmacokinetic Assay - Bridging Format

Pharmacokinetic (PK) Assay - Bridging 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

PK bridging ELISA using antibodies HCA182 and HCA184P

Figure 1: PK bridging ELISA using antibodies HCA182 and HCA184P

Human anti-bevacizumab antibodies, product codes HCA182 and HCA184P

Figure 1: Anti-bevacizumab, clone AbD17976 (HCA182) was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, bevacizumab was spiked in the given concentrations into 10% human serum. Detection was performed by HRP conjugated anti-bevacizumab, clone AbD (HCA184P) at a concentration of 4.0 µg/ml in HISPEC buffer (BUF049). QuantaBlu™ fluorogenic peroxidase was used as the substrate.

Protocol: PK bridging ELISA for use with anti-bevacizumab antibodies


Anti-Drug Antibody Assay - Bridging ELISA

Immunogenicity Assay – Bridging Format

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: Immunogenicity assay, bridging ELISA format

Figure 2: ADA assay, bridging ELISA format

Human anti-bevacizumab antibody, product code HCA185

Figure 2: Bevacizumab was coated at 1.0 µg/ml on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, anti-bevacizumab antibody clone AbD17976_hIgG1 (HCA185) was spiked in the given concentrations into 10% human serum. Detection was performed by adding HRP conjugated bevacizumab at a concentration of 2.0 µg/ml in HISPEC buffer (BUF049).  QuantaBlu fluorogenic peroxidase was used as the substrate.

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


Inhibition ELISA - Inhibition of bevacizumab binding to human VEGF

Schematic image of inhibition assay. Drug target capture antigen (red), monoclonal antibody drug (gold), anti-IgG Fc specific detection antibody (gray) labeled with HRP

Figure 3: Inhibition of bevacizumab to human VEGF by antibody HCA182

Figure 3: Inhibition of bevacizumab to human VEGF by antibody HCA182

Human anti-bevacizumab antibody, product code HCA182

Figure 3: A microtiter plate was coated over night with human VEGF at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, a pre-incubated mixture of bevacizumab (0.3 µg/ml) plus increasing concentrations of human anti-bevacizumab antibody clone AbD17976 (HCA182) in monovalent Fab format was added. Free bevacizumab, still capable of binding to the human VEGF-coated plate, was detected using HRP conjugated mouse anti human IgG (Fc) CH2 domain specific antibody (MCA647P) and QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements. 


Determination of Antibody Specificity

Specificity of anti-bevacizumab antibody HCA182
Figure 4: Specificity of anti-bevacizumab antibody HCA182

Human anti-bevacizumab antibody, product code HCA182

Figure 4: Demonstration of the specificity of antibodies to bevacizumab. Antigens were coated at 5 µg/ml on a microtiter plate over night. After washing and blocking with 5% BSA, 20µl of anti-bevacizumab antibody, clone AbD17976 (HCA182) from a 2 µg/ml solution in PBST buffer, was added. Detection was performed using HRP conjugated anti-Strep-tag secondary antibody (MCA2489P), diluted 1:5000 in HISPEC buffer (BUF049). QuantaBlu fluorogenic peroxidase was used as the 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.

Avastin® is a registered trademark of Genentech, Inc. HuCAL® is a registered trademark of MorphoSys AG. QuantaBlu™ is a trademark of Thermo Fisher Scientific.