Anti-Denosumab Antibodies

Antibodies for bioanalytical and patient monitoring assays for denosumab and biosimilar products

Recombinant monoclonal anti-idiotypic antibodies, highly specific for the fully human IgG2 kappa antibody drug denosumab (Prolia, Xgeva)

Type 1 anti-idiotypic antibody

Anti-Denosumab Inhibitory Antibodies

These antibodies inhibit the binding of the drug denosumab to its target, RANKL (receptor activator of nuclear factor kappa-B ligand), and therefore detect free drug. A pair of these antibodies is ideal for development of a pharmacokinetic (PK) bridging ELISA. Three antibodies of high, medium or low affinity are suitable as a reference standard for an anti-drug antibody (ADA) assay.

Catalog Numbers: HCA280, HCA280P, HCA281, HCA282, HCA288

Anti-Denosumab Non-Inhibitory Antibody

Anti-Denosumab Non-Inhibitory Antibody

This non-inhibitory antibody does not inhibit the binding of denosumab to RANKL and can therefore detect total drug – free, partially bound and fully bound. This antibody can be used in conjunction with an inhibitory antibody in a PK bridging ELISA to measure free drug.

Catalog Numbers: HCA283, HCA283P

Our recombinant, monoclonal antibodies are generated using the Human Combinatorial Antibody Library (HuCAL®) 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. These fully human antibodies are also suitable 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-biotherapeutic antibodies and their binding types and properties

Anti-biotherapeutic antibody quality control and characterization

Table 1: Antibodies Specific to Denosumab.

Catalog #

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA288

AbD26296

Denosumab

Inhibitory

Fab-FH1

7.1

PK bridging ELISA with HCA280P

HCA280

AbD26295_hIgG1

Denosumab

Inhibitory

Human
IgG1

1.8*

PK bridging ELISA with HCA288
ADA assay

HCA280P

AbD26295_hIgG1

Denosumab

Inhibitory

Human
IgG1
HRP labeled

1.8*

PK bridging ELISA with HCA288

HCA281

AbD26296_hIgG1

Denosumab

Inhibitory

Human
IgG1

7.1*

ADA assay

HCA282

AbD26862_hIgG1

Denosumab

Inhibitory

Human
IgG1

0.8*

ADA assay

HCA283

AbD26781_hIgG1

Denosumab & Denosumab/RANKL complex

Non-inhibitory

Human IgG1

19* (Denosumab)
17* (Denosumab/RANKL complex)

PK bridging ELISA detection, with HCA280 or HCA282 capture

HCA283P

AbD26781_hIgG1

Denosumab & Denosumab/RANKL complex

Non-inhibitory

Human IgG1 HRP labeled

19* (Denosumab)
17* (Denosumab/RANKL complex)

PK bridging ELISA detection, with HCA280 or HCA282 capture

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

Related Products

Denosumab drug isotype control Recombinant Human IgG2 Kappa (HCA193)

Recommended secondary antibodies for Fab detection tags


Pharmacokinetic Assay

Schematic image of anti-idiotypic antibodies in PK bridging ELISA format

Schematic image of PK bridging ELISA. Anti-idiotypic capture antibody, Ig format (blue), monoclonal antibody drug (gold), anti-idiotypic detection antibody, Ig format, labeled with HRP.

Fig. 1. Denosumab PK ELISA bridging format using antibodies HCA288 and HCA280P.

Fig. 1. Denosumab PK ELISA bridging format using antibodies HCA280 or HCA282 and HCA283P.

Measurement of free drug with Human Anti-Denosumab Antibodies HCA280 or HCA282 and HCA283P

In Figure 1, Human Anti-Denosumab Antibody, clone AbD26295_hIgG1 (HCA280) or clone AbD26862_hIgG1 (HCA282) 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% human serum was added, spiked with increasing concentrations of denosumab. Detection was performed using HRP conjugated Human Anti-Denosumab Antibody, clone AbD26781_hIgG1 (HCA283P) diluted to 2 µg/ml in HISPEC Assay Diluent (BUF049A), plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of four measurements.

Protocol: PK bridging ELISA protocol denosumab HCA280 or HCA282 and HCA283P


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 HCA280, HCA281 and HCA282.

Fig. 2. ADA bridging ELISA using antibodies HCA280, HCA281 and HCA282.

Human Anti-Denosumab Antibodies HCA280, HCA281 and HCA282
In Figure 2, denosumab 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 Human Anti-Denosumab Antibody, clones AbD26295_hIgG1 (HCA280), AbD26296_hIgG1 (HCA281) or AbD26862_hIgG1 (HCA282). Detection was performed using HRP conjugated denosumab at 2 µg/ml in HISPEC Assay Diluent, and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. HRP conjugation of denosumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit®.

Protocol: ADA bridging ELISA protocol denosumab HCA280, HCA281, HCA282


Inhibition ELISA

Fig. 3. Demonstration of inhibitory properties of antibodies HCA280, HCA282 and HCA288.

Fig. 3. Demonstration of inhibitory properties of antibodies HCA280, HCA282, HCA283 and HCA288.


Human Anti-Denosumab Antibodies HCA280, HCA282, HCA283 and HCA288

In Figure 3, Human RANKL was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, denosumab was added (0.3 µg/ml), spiked with increasing concentrations of antibodies HCA280, HCA282 and HCA283 (all in monovalent Fab format), and HCA288. Free denosumab, still capable of binding to the RANKL 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. It should be noted that in this assay set-up, the non-inhibitory antibody HCA283 shows a slight decrease in signal. This is most likely due to different binding modes of the denosumab-RANKL complex at higher denosumab concentrations (Arthur et al. 2012).


Demonstration of Antibody Specificity

Fig. 4. Demonstration of the specificity of antibody HCA288 to denosumab.

Fig. 4. Demonstration of the specificity of antibody HCA288 to denosumab.

Human Anti-Denosumab Antibody HCA288
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-Denosumab Antibody, clone AbD26296 (HCA288) at a concentration of 2 μg/ml and an HRP conjugated anti-DYKDDDDK tag antibody in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.

Fig. 5. Demonstration of the specificity of antibody HCA283 to denosumab and RANKL/denosumab complex.

Fig. 5. Demonstration of the specificity of antibody HCA283 to denosumab and RANKL/denosumab complex.

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-Denosumab Antibody clone AbD26781_hIgG1 (HCA283P) at a concentration of 2 μg/ml in HISPEC Assay Diluent (BUF049A) and QuantaBlu Fluorogenic Peroxidase Substrate.


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