Anti-Etanercept Antibodies

Antibodies for bioanalytical assays to measure etanercept and biosimilar products

Recombinant monoclonal antibodies, highly specific for the biotherapeutic fusion protein etanercept (Enbrel, Etacept, Intacept). Etanercept is a dimeric fusion protein consisting of the extracellular ligand binding portion of the human 75 kDa (p75) tumor necrosis factor receptor (TNFR2) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1.

Anti-etanercept p75-specific inhibitory antibody

Anti-etanercept p75-specific inhibitory antibody

The anti-etanercept inhibitory antibody blocks the binding of etanercept to its target, tumor necrosis factor alpha (TNFα). The antibody does not bind to etanercept when it is bound to TNFα. This antibody is ideal for use in direct or indirect ELISA, in a bridging ELISA to develop pharmacokinetic (PK) assays to detect free drug, or as a reference standard for anti-drug antibody (ADA) assays. Catalog Numbers: HCA277, HCA277P
 

Anti-etanercept p75-specific non-inhibitory antibody

Anti-etanercept p75-specific non-inhibitory antibody

The anti-etanercept non-inhibitory antibody binds to the p75 subunit of etanercept, but does not inhibit the binding of TNFα. It binds to both free etanercept and to etanercept bound to TNFα and can therefore be used in a bridging ELISA to develop PK assays to detect total drug (free, bound or partially bound).
Catalog Number:  HCA276

 

Anti-etanercept fusion-specific antibody Anti-etanercept fusion-specific antibody

This antibody specifically recognizes etanercept at the region of the fusion of p75 with the Fc portion of human IgG1. It binds to both free etanercept and to etanercept bound to TNFα. It does not bind to p75 (soluble TNFR2) or to human IgG1. It does not inhibit the binding of etanercept to TNFα. This product may be used in a direct or indirect ELISA or as a detection antibody in a bridging ELISA to measure total etanercept very specifically.
Catalog Numbers: HCA279, HCA279P

These recombinant, monoclonal antibodies are generated using the HuCAL® Combinatorial Antibody Library and CysDisplay®, a proprietary method of phage display, with guided selection methods to obtain highly targeted reagents. The result is highly specific and high affinity antibodies, ideal for development of PK assays. Because these antibodies are fully human, they are also perfect as controls or calibrators for ADA assays. 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 Etanercept

Catalog Number

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA276

AbD25939

Etanercept (p75 TNFR2)

Non-inhibitory

Fab-FH1

3.2

PK bridging ELISA with HCA277P or HCA279P

HCA277

AbD25940_hIgG4Pro

Etanercept (p75 TNFR2)

Inhibitory

Human
IgG4Pro2

2.4*

PK bridging ELISA with HCA276
ADA assay

HCA277P

AbD25940_hIgG4Pro

Etanercept (p75 TNFR2)

Inhibitory

Human
IgG4Pro2
HRP labeled

2.4*

PK bridging ELISA with HCA276

HCA279

AbD26183_hIgG1

Etanercept

Fusion-specific, non-inhibitory

Human
IgG1

14.8*

PK ELISA with HCA276

HCA279P

AbD26183_hIgG1

Etanercept

Fusion-specific, non-inhibitory

Human
IgG1
HRP labeled

14.8*

PK ELISA with HCA276

Table 1 Antibody Specifications

1 F=DYKDDDDK-tag, H=His-6-tag
2 IgG4 isotype with a mutation in the core hinge region that prevents the formation of IgG4 half molecules
*Affinity measured in the monovalent Fab form


Pharmacokinetic Assays

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

Fig. 1. Etanercept PK ELISA bridging format using antibodies HCA276 and HCA277

Fig. 1. Etanercept PK ELISA bridging format using antibodies HCA276 and HCA277P


Measurement of free drug with Human Anti-Etanercept Antibodies HCA276 and HCA277P

In Figure 1, Anti-Etanercept Antibody clone AbD25939 (HCA276) was coated on a microtiter plate at 5 µg/ml and left overnight. Washing and blocking was performed with PBST + 5% BSA. 10% human serum was added spiked with increasing concentrations of etanercept. Detection was performed using HRP conjugated Anti-Etanercept Antibody clone AbD25940_hIgG4Pro (HCA277P) diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049A), plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: PK bridging ELISA protocol etanercept HCA276 and HCA277P

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

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

Fig. 2. Etanercept PK ELISA using antibodies HCA276 and HCA279

Fig. 2. Etanercept PK ELISA using antibodies HCA276 and HCA279P


Measurement of total drug with Human Anti-Etanercept Antibodies HCA276 and HCA279P

In Figure 2, Anti-Etanercept Antibody, clone AbD25939 (HCA276) was coated on a microtiter plate at 5 µg/ml and left overnight. Washing and blocking was performed with PBST + 5% BSA. 10% human serum was added spiked with increasing concentrations of etanercept. Detection was performed using HRP conjugated Anti-Etanercept Antibody clone AbD26183_hIgG1 (HCA279P) diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049A), plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.

Protocol: PK bridging ELISA protocol etanercept HCA276 and HCA279P


Anti-Drug Antibody Assay – Bridging ELISA

Schematic image of ADA bridging assay. Fusion protein drug as capture and detection reagent labeled with HRP (gold-green), fully human anti-drug antibody, Ig format (blue).

Schematic image of ADA bridging assay. Fusion protein drug as capture and detection reagent labeled with HRP (gold-green), fully human anti-drug antibody, Ig format (blue).

Fig. 3. ADA assay, bridging format using anti-etanercept antibody HCA277

Fig. 3. ADA assay, bridging format using anti-etanercept antibody HCA277


Human Anti-Etanercept Antibody HCA277

In Figure 3, etanercept was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with PBST + 5% BSA, PBST with 10% human serum was added spiked with increasing concentrations of the human Anti-Etanercept Antibody clone AbD25940_hIgG4Pro (HCA277). Detection was performed using HRP conjugated etanercept at 2 µg/ml in HISPEC Assay Diluent (BUF049A), and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. HRP conjugation of etanercept was performed using a LYNX Rapid HRP Antibody Conjugation Kit.

Protocol: ADA bridging ELISA protocol etanercept HCA277


Inhibition ELISA

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

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

Fig. 4. Demonstration of inhibitory and non-inhibitory properties of anti-etanercept antibodies HCA277 and HCA279

Fig. 4. Demonstration of inhibitory and non-inhibitory properties of anti-etanercept antibodies HCA277 and HCA279


Human Anti-Etanercept Antibodies HCA277 and HCA279

In Figure 4,  Recombinant Human TNFα (PHP051) was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, a pre-incubated mixture of etanercept (0.3 µg/ml) plus increasing concentrations of Anti-Etanercept Antibody clone AbD25940 (HCA277, inhibitory antibody), or clone AbD26183 (HCA279, non-inhibitory p75-Fc fusion specific antibody) in the monovalent Fab format was added. Free etanercept, still capable of binding to the human TNFα coated plate, was detected using Mouse Anti-Human IgG (Fc) CH2 Domain:HRP (MCA647P) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.


Determination of Antibody Specificity

Fig. 5. Demonstration of the specificity of antibody HCA279 to etanercept
Fig. 5. Demonstration of the specificity of antibody HCA279P to etanercept

Human Anti-Etanercept Antibody HCA279P

In Figure 5, 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 Anti-Etanercept Antibody clone AbD26183_hIgG1 (HCA279P) at a concentration of 2 μg/ml in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate. The data shows that the antibody detects etanercept fusion protein specifically, and not human IgG1 or human TNFR2.


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.