Anti-Nivolumab Antibodies

Antibodies for bioanalytical and patient drug level monitoring assays for nivolumab

Recombinant monoclonal anti-idiotypic antibodies, highly specific for the immune checkpoint inhibitor drug nivolumab (Opdivo).

Anti-Nivolumab Inhibitory Antibodies

Anti-Nivolumab Inhibitory Antibodies

These antibodies inhibit the binding of the drug nivolumab to its target, programmed cell death 1 (PD-1), and therefore detect free drug. An antibody pair is ideal for the development of a pharmacokinetic (PK) bridging ELISA. Four antibodies of high, medium or low affinity are suitable as a reference standard for an anti-drug antibody (ADA) assay.

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 Nivolumab.

Catalog #

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA299

AbD30255

Nivolumab

Inhibitory

Fab-FH1

0.5

PK bridging ELISA capture antibody

HCA300

AbD30255_hIgG1

Nivolumab

Inhibitory

Human IgG1

0.52

ADA assay

HCA301

AbD30258_hIgG1

Nivolumab

Inhibitory

Human IgG1

22

PK bridging ELISA detection antibody

ADA assay

HCA301P
coming soon

AbD30258_hIgG1

Nivolumab

Inhibitory

Human IgG1
HRP labeled

22

PK bridging ELISA detection antibody

HCA302

AbD30260_hIgG1

Nivolumab

Inhibitory

Human IgG1

232

ADA assay

HCA303

AbD30264_hIgG1

Nivolumab

Inhibitory

Human IgG1

142

ADA assay

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

Related Product

Nivolumab drug isotype control Recombinant Human IgG4 Kappa (HCA247)


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. Nivolumab PK ELISA bridging format using antibodies HCA299 and HCA301P

Fig. 1. Nivolumab PK ELISA bridging format using antibodies HCA299 and HCA301P.

Measurement of free drug with human anti-nivolumab antibodies

In Figure 1, Human Anti-Nivolumab Antibody, clone AbD30255 (HCA299) 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, and spiked with increasing concentrations of nivolumab was added. Detection was performed using HRP conjugated Human Anti-Nivolumab Antibody, clone AbD30258_hIgG1 (HCA301P) 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: Nivolumab PK bridging ELISA protocol HCA299 and HCA301P


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 HCA300, HCA301, HCA302 and HCA303.

Fig. 2. ADA bridging ELISA using antibodies HCA300, HCA301, HCA302 and HCA303.

Immunogenicity assay using human anti-nivolumab antibodies as calibration standard

In Figure 2, nivolumab 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-Nivolumab Antibody, clones AbD30255_hIgG1 (HCA300), AbD30258_hIgG1 (HCA301), AbD30260_hIgG1 (HCA302) or AbD30264_hIgG1 (HCA303). Detection was performed using HRP conjugated nivolumab 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 nivolumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit®.

Protocol: Nivolumab ADA bridging ELISA protocol


Inhibition ELISA

Fig. 3. Demonstration of the inhibitory property of antibody HCA301.

Fig. 3. Demonstration of the inhibitory property of antibody HCA301.


In Figure 3, recombinant human PD-1 was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, nivolumab was added (0.3 µg/ml), spiked with increasing concentrations of Human Anti-Nivolumab Antibody clone AbD30258 (HCA301) in monovalent Fab format. Free Nivolumab, still capable of binding to the PD-1 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

Fig. 4. Specificity of Human Anti-Nivolumab Antibody HC301.
Fig. 4. Specificity of Human Anti-Nivolumab Antibody HC301.

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-Nivolumab Antibody, clone AbD30258_hIgG1 (HCA301P) 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.