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 Inhibitory Antibodies

The inhibitory (Type 1) anti-bevacizumab antibodies detect free drug. These antibodies are ideal for use in direct ELISA, pharmacokinetic (PK) bridging assays and as a positive control or calibrator for immunogenicity 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 Human Combinatorial Antibody Library (HuCAL®) technology and CysDisplay®, a proprietary method of phage display, which results in highly specific and sensitive reagents, ideal for the development of bioanalytical ligand binding assays. Because these antibodies are fully human, they are also ideal as a surrogate positive control or calibrator in 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

Ranibizumab3

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
3 Ranibizumab originates from the same parental mouse antibody as bevacizumab; it has 4 amino acid exchanges in the heavy chain and 1 amino acid exchange in the light chain compared to bevacizumab

Related Products

Bevacizumab drug isotype control Recombinant human IgG1 kappa (HCA192)
Bevacizumab biosimilar for research use Human Anti-VEGF Antibody (MCA6089) 
Bevacizumab drug target Recombinant Human VEGF-A (PHP293)


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

Fig. 1. PK bridging ELISA using antibodies HCA182 and HCA184P.

Measurement of free drug with Anti-Bevacizumab Antibodies

In Figure 1, Anti-Bevacizumab Antibody 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 using HRP conjugated Anti-Bevacizumab Antibody clone AbD16748_hIgG1 (HCA184P) at a concentration of 4.0 µg/ml in HISPEC Assay Diltuent (BUF049), and QuantaBlu Fluorogenic Peroxidase Substrate.

Protocol: Bevacizumab PK bridging ELISA


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

Fig. 2. Bevacizumab ADA bridging ELISA using HCA185.

Bevacizumab ADA assay using HCA185 as calibration standard

In 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 Assay Diluent (BUF049), and QuantaBlu Fluorogenic Peroxidase Substrate.

Protocol: Bevacizumab ADA bridging ELISA for use with Anti-Bevacizumab Antibody HCA185

Schematic image of PK Bridging ELISA. Capture antibody, Fab format (purple), fusion protein drug (gold-green), detection antibody, Ig format (blue), labeled with HRP

Schematic image of ADA bridging assay. Monoclonal Fab fragment antibody drug as capture antibody and detection antibody labeled with HRP (gold), fully human anti-idiotypic antibody, Ig format (blue).

Fig. 3. Ranibizumab ADA bridging ELISA using HCA185.

Fig. 3. Ranibizumab ADA bridging ELISA using HCA185.


Ranibizumab ADA assay using HCA185 as calibration standard

In Figure 3, a microtiter plate was coated overnight with ranibizumab at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, 10% human serum was added spiked with increasing concentrations of Human Anti-Bevacizumab Antibody clone AbD17976_hIgG1 (HCA185). Detection was performed using HRP conjugated ranibizumab at a concentration of 2 µg/ml in HISPEC Assay Diluent (BUF049A) followed by QuantaBlu Fluorogenic Peroxidase Substrate.

Protocol: Ranibizumab ADA bridging ELISA for use with Anti-Bevacizumab Antibody HCA185


Inhibition ELISA

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

Fig. 4. Demonstration of the inhibitory property of antibody HCA182.


In Figure 4, a microtiter plate was coated over night with Human Recombinant VEGF-A (PHP293) 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 VEGF-coated plate, was detected using HRP conjugated Mouse Anti-Human IgG (Fc) CH2 Domain Antibody (MCA647P) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.


Demonstration of Antibody Specificity

Specificity of anti-bevacizumab antibody HCA182
Fig. 5. Specificity of Anti-Bevacizumab Antibody HCA182.

In Figure 5, 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 Mouse Anti-Strep-Tag Classic Antibody (MCA2489P), diluted 1:5000 in HISPEC Assay Diluent (BUF049), and 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.