Anti-Rituximab Antibodies

Antibodies for bioanalytical and clinical monitoring assays for rituximab and biosimilar products

Four recombinant monoclonal anti-idiotypic antibodies, and one rat monoclonal antibody, highly specific for the chimeric mouse/human monoclonal antibody drug rituximab (Rituxan, MabThera).

Type 1 anti-idiotypic antibody

Anti-Rituximab Inhibitory Antibodies

All antibodies inhibit the binding of the drug rituximab to its target CD20, and therefore detect free drug. A pair of these antibodies is ideal for development of a pharmacokinetic (PK) bridging ELISA. One antibody, in fully human IgG1 format, is 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. A fully human antibody is also suitable 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 on anti-biotherapeutic antibodies and their binding types and properties

Anti-biotherapeutic antibody quality control and characterization

Table 1: Antibodies specific to Rituximab.

Catalog #

Clone

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

MCA2260

MB2 A4

Inhibitory

Rat IgG2a

Not determined

PK bridging ELISA detection, with HCA186

MCA2260F

MB2 A4

Inhibitory

Rat IgG2a
FITC labeled

Not determined

Flow cytometry

MCA2260P

MB2 A4

Inhibitory

Rat IgG2a
HRP labeled

Not determined

PK bridging ELISA detection, with HCA186

HCA061

AbD02842

Inhibitory

Fab-dHLX-MH1

4.9*

Indirect ELISA

HCA061P

AbD02842

Inhibitory

Fab-dHLX-MH
HRP labeled

4.9*

PK bridging ELISA, detection antibody

HCA062

AbD02844

Inhibitory

Fab-dHLX-MH

10.0*

Indirect ELISA

HCA062P

AbD02844

Inhibitory

Fab-dHLX-MH
HRP labeled

10.0*

PK bridging ELISA, detection

HCA186

AbD18423

Inhibitory

Fab-FH2

0.13

PK bridging ELISA capture, with MCA2260

HCA201

AbD18423_hIgG1

Inhibitory

Human IgG1

0.13*

ADA assay

1 dHLX=double helix loop helix motif dimerization domain M=myc-tag H=His-6-tag
2 F=DYKDDDDK-tag H=His-6-tag
*Affinity measured in the monovalent Fab format

Related Product

Rituximab biosimilar for research use Human Anti-CD20 Antibody (MCA6091)


Pharmacokinetic Assay - Bridging ELISA

Pharmacokinetic (PK) Assay

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 assay, bridging format, using rituximab antibody

Fig.1. PK assay, bridging format, using, antibodies HCA186 and MCA2260P


Measurement of free drug with Anti-Rituximab Antibodies HCA186 and MCA2260P

In Figure 1, Human Anti-Rituximab Antibody, clone AbD18432 (HCA186) was coated on a microtiter plate at 1.0 µg/ml and left overnight. After washing and blocking with 5% BSA in PBST, rituximab spiked in 10% human serum was added in the given concentrations. Detection was performed using HRP conjugated Rat Anti-Rituximab Antibody, clone MB2 A4 (MCA2260P), at 3.0 µg/ml in HISPEC Assay Diluent (BUF049), plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three experiments.

Protocol: PK bridging ELISA protocol rituximab with HCA186 and MCA2260P


Anti-Drug Antibody Assay – Bridging ELISA

Anti-Drug Antibody (ADA) Assay – Bridging ELISA

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)

ADA assay, bridging, format using Rituximab antibody

Fig.2. ADA assay, bridging, format using antibody HCA201


Human Anti-Rituximab Antibody HCA201

In Figure 2, Rituximab was coated at two different concentrations (1.0 µg/ml and 1.5 µg/ml) on a microtiter plate and left overnight. Anti-Rituximab Antibody, clone AbD18423_hIgG1 (HCA201), titrated into 10% human serum in the given concentrations, was added. Detection was performed using HRP conjugated Rituximab in HISPEC Assay Diluent, plus QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three experiments.


Determination of Antibody Specificity

Specificity of anti-rituximab antibody HCA186
Fig.3. Specificity of Anti-Rituximab Antibody HCA186

Anti-Rituximab Antibody HCA186

In Figure 3, various antigens (human serum, chimeric, humanized and human antibodies of the type IgG1/kappa) were coated at 5.0 µg/ml on a microtiter plate and left overnight. After washing and blocking, Anti-Rituximab Antibody, clone AbD18423 (HCA186) was added. Detection was performed using Mouse Anti-Penta Histidine Tag:HRP Antibody (MCA5995P).


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.