Antibodies for bioanalytical method development to measure natalizumab and biosimilar products
Ready-made recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized monoclonal antibody drug natalizumab (Tysabri®).
The Type 1 anti-natalizumab antibodies inhibit the binding of the humanized monoclonal antibody drug natalizumab to its target, human alpha4-beta1-integrin, and therefore detect free drug. These antibodies are ideal for use in direct and indirect ELISA, to develop pharmacokinetic (PK) bridging assays and as a reference standard for anti-drug antibody (ADA) assays.
Product codes: HCA248, HCA249, HCA249P, HCA250
These recombinant monoclonal antibodies are generated using the HuCAL® antibody library and an improved method of phage display, which results in highly specific and sensitive reagents. These are ideal for PK and PD assays. Antibodies are fully human, so they are also perfect as reference standards for ADA assays. The in vitro production of recombinant antibodies means that there is a consistent, secure supply throughout preclinical development and clinical trials, avoiding the need to develop new reagents.
More information about anti-idiotypic antibodies and their binding types and properties
|Product Code||Clone||Antibody Specificity||Binding Type||Format||Affinity KD, nM||Assay Development Recommendations|
PK bridging ELISA, capture antibody with HCA249
|HCA249||AbD21375_hlgG1||Natalizumab||Type 1||Human lgG1||2.12||
PK bridging ELISA, detection antibody with HCA248
ADA assay, high affinity
PK bridging ELISA, detection antibody with HCA248
|HCA250||AbD21384_hlgG1||Natalizumab||Type 1||Human lgG1||12.22||
ADA assay, medium affinity
Table 1 Antibody Specifications
1 F=DYKDDDDK-tag; H=His-6-tag
2 Affinity measured in the monovalent Fab form
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
Figure 1: PK assay, bridging format, using antibodies HCA248 and HCA249
Figure 1: A microtiter plate was coated over night with anti-natalizumab antibody, clone AbD21375 (HCA248) at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, 10% human serum was added spiked with increasing concentrations of natalizumab. Detection was performed using HRP conjugated anti-natalizumab antibody, clone AbD21375_hIgG1 (HCA249P) at a concentration of 2 µg/ml in HISPEC assay diluent (BUF049A) and QuantaBlu™ fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
Schematic image of inhibition assay. Drug target capture antigen (red), monoclonal antibody drug (gold), anti-IgG Fc specific detection antibody (gray) labeled with HRP
Figure 2: Inhibition of natalizumab binding to human alpha4-beta1-integrin by HCA248
Figure 2: A microtiter plate was coated over night with human alpha4-beta1-integrin at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, a pre-incubated mixture of natalizumab (0.3 µg/ml) plus increasing concentrations of human anti-natalizumab antibody clone AbD21375 (HCA248) in the monovalent Fab format was added. Free natalizumab, still capable of binding to the human alpha4-beta1-integrin coated plate, was detected using HRP conjugated mouse anti human IgG (Fc) CH2 domain (MCA647P) and QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
Two anti-natalizumab antibodies in IgG1 format are available, each with a different affinity for the target (Table 1), allowing the ADA assay to be tailored to the specific needs of the developer.
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)
Figure 3: ADA bridging ELISA with HCA249 or HCA250
Figure 3: A microtiter plate was coated over night with natalizumab at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, PBST with 10% human serum was added spiked with increasing concentrations of human anti-natalizumab antibody, either HCA249 or HCA250. Detection was performed using HRP conjugated natalizumab*. Data are shown as the mean of three measurements.
Figure 4: Specificity of anti-natalizumab antibody HCA248
Figure 4: A microtiter plate was coated over night with various antigens at a concentration of 5 μg/ml. After washing and blocking with PBST+5% BSA, detection was performed using human anti-natalizumab antibody clone AbD21375 (HCA248) at a concentration of 2 μg/ml followed by HRP conjugated rat anti-DYKDDDDK tag antibody (MCA4764P, 1:500 dilution) in HISPEC assay diluent (BUF049A) and QuantaBlu fluorogenic peroxidase substrate.
* Antibody conjugated to HRP using a LYNX Rapid Conjugation Kit® (product codes LNK001P - LNK006P)
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.
HuCAL® is a registered trademark of MorphoSys AG. QuantaBlu™ is a trademark of Thermo FisherScientific. Tysabri® is a registered trademark of Elan Pharmaceuticals, Inc.