Anti-Eculizumab Antibodies

Antibodies for drug level monitoring assays and bioanalytical assays for biosimilar development

Recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized IgG2/4 kappa monoclonal antibody drug eculizumab (Soliris).

 Anti-Vedolizumab Inhibitory Antibodies

Anti-Eculizumab Inhibitory Antibodies

These four antibodies inhibit the binding of the drug eculizumab to its target, complement C5 protein, and therefore detect free drug. An antibody pair is ideal for the development of a pharmacokinetic (PK) bridging ELISA. Three antibodies of high, medium and lower affinity are suitable as a positive control for an anti-drug antibody (ADA) assay.

Catalog Numbers: HCA312, HCA313, HCA313P, HCA314, HCA315

These recombinant, monoclonal antibodies are generated using Human Combinatorial Antibody Library (HuCAL®) technology and CysDisplay®, a proprietary method of phage display, and use guided selection methods to obtain highly targeted reagents. The result is highly specific and high affinity antibodies, ideal for development of PK assays. HuCAL antibodies are fully human and, when in full length IgG format, can be used as a control or calibrator in an ADA assay. 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 antibody binding types and their properties

Anti-biotherapeutic antibody quality control and characterization

Table 1. Antibodies Specific to Eculizumab.

Catalog #

 

Clone

 

Antibody Specificity

 

Binding Type

 

Format

 

Affinity

KD, nM

 

Assay Development Recommendations

HCA312

AbD32311

Eculizumab

Inhibitory

Fab-FH1

6

PK bridging ELISA capture antibody

HCA313

AbD32107_hIgG1

Eculizumab

Inhibitory

Human IgG1

0.62

PK bridging ELISA detection antibody

ADA assay

HCA313P

AbD32107_hIgG1

Eculizumab

Inhibitory

Human IgG1

HRP labeled

0.62

PK bridging ELISA detection antibody

HCA314

AbD32311_hIgG1

Eculizumab

Inhibitory

Human IgG1

62

ADA assay

HCA315

AbD32109_hIgG1

Eculizumab

Inhibitory

Human IgG1

32

ADA assay

1 F=DYKDDDDK-tag H=His-6-tag
2 Affinity measured in the monovalent Fab format

Related Product

Eculizumab biosimilar for research use Human Anti-C5 Antibody (MCA6103) 


Pharmacokinetic Assay

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, Ig format (blue), labeled with HRP.

Fig. 1. Eculizumab PK ELISA bridging format using antibodies HCA312 and HCA313P.

Fig. 1. Eculizumab PK ELISA bridging format using antibodies HCA312 and HCA313P.

Measurement of free drug with Human Anti-Eculizumab Antibodies

In Figure 1, Human Anti-Eculizumab Antibody clone AbD32311 (HCA312) was coated on a microtiter plate at 1 µg/ml and left overnight. Washing and blocking was performed with PBST + 5% BSA. PBST with 10% fetal calf serum, spiked with increasing concentrations of eculizumab was added. Detection was performed using HRP conjugated Human Anti-Eculizumab Antibody, clone AbD32107_hIgG1 (HCA313P) diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Eculizumab PK bridging ELISA protocol HCA312 and HCA313P 


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).

Fig. 2. ADA bridging ELISA using antibodies HCA313, HCA314 and HCA315.

Fig. 2. ADA bridging ELISA using antibodies HCA313, HCA314 and HCA315.

Immunogenicity assay using Human Anti-Eculizumab Antibodies as the calibration standard

In Figure 2, eculizumab was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with PBST + 5% BSA, PBST with 10% fetal calf serum was added, spiked with increasing concentrations of Human Anti-Eculizumab Antibody, clone AbD32107_hIgG1 (HCA313, black), clone AbD32311_hIgG1 (HCA314, red), or clone AbD32109_hIgG1 (HCA315, blue). Detection was performed using HRP conjugated eculizumab at 2 µg/ml in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: Eculizumab ADA bridging ELISA


Inhibition ELISA

Fig. 3. Demonstration of the inhibitory property of antibody HCA313.

Fig. 3. Demonstration of the inhibitory property of antibody HCA313.


In figure 3, human complement C5 was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, eculizumab was added (0.3 µg/ml), spiked with increasing concentrations of Human Anti-Eculizumab Antibody clone AbD32107 (HCA313) in the monovalent Fab format. Free eculizumab still capable of binding to the human complement C5 coated plate, was detected using Mouse Anti-Human IgG (Fc) CH2 Domain:HRP Antibody (MCA647P), followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.


Demonstration of Antibody Specificity
 

Specificity of Human Anti-Eculizumab Antibody HC314.
Fig. 4. Specificity of Human Anti-Eculizumab Antibody HC314.

In Figure 4, a microtiter plate was coated overnight with various antigens at a concentration of  5 µg/ml. After washing and blocking with PBST + 5% BSA, detection was performed using HRP conjugated Human Anti-Eculizumab Antibody, clone AbD32311_hIgG1 (HCA314) at a concentration of 2 μg/ml in HISPEC Assay Diluent (BUF049A), 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.