Highly specific and sensitive reagents for antibody drug development assays.
Technical articles featuring applications of anti-idiotypic antibodies.
Ready-made recombinant, anti-idiotypic monoclonal antibodies, highly specific for the human antibody drug ustekinumab (STELARA®)
The Type 1 anti-ustekinumab antibodies inhibit the binding of the drug ustekinumab to its target, IL-12 and IL-23, and therefore detect free drug. These antibodies are ideal for use in direct ELISA, pharmacokinetic (PK) bridging assays and to develop and calibrate immune response (IR) assays to measure the anti-drug antibody (ADA) response in patient sera.
The recombinant anti-ustekinumab antibodies are generated using the HuCAL® antibody library and a modified and improved form of phage display. Antibody generation using HuCAL technology results in highly specific and sensitive antibodies, which are ideal for PK and PD assays. Antibodies are fully human, so are also suitable as controls or calibrators 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|
|HCA208||AbD17829||Ustekinumab||Type 1||Fab-V5Sx21||2.8||PK bridging ELISA capture antibody with HCA210|
|HCA209||AbD18334||Ustekinumab||Type 1||Fab-FH2||0.4||PK bridging ELISA capture antibody with HCA210|
|HCA210||AbD17827_hIgG1||Ustekinumab||Type 1||Human IgG1||0.2*||
PK bridging ELISA detection antibody
|0.2*||PK bridging ELISA detection antibody|
|HCA211||AbD17829_hIgG1||Ustekinumab||Type 1||Human IgG1||2.8*||ADA assay|
Table 1: Antibody Specifications
1 Fab antibody; V5-tag; StrepX-StrepX-tag
2 F=DYKDDDDK-tag; H=His-6-tag
*Affinity measured in the monovalent Fab format
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 bridging ELISA using antibodies HCA209 and HCA210
Figure 1: Anti-ustekinumab antibody, clone AbD18334 (HCA209), was coated at 1.0 µg/ml on a microtiter plate and left over night. Washing and blocking was performed with 5% BSA in PBST. Ustekinumab, titrated into 10% human serum, was then added. Detection was performed by adding HRP conjugated anti-ustekinumab antibody, clone AbD17827_hIgG1 (HCA210P), 2 µg/ml in HISPEC assay diluent (BUF049), plus QuantaBlu™ fluorogenic peroxidase substrate. The data are shown as the mean of three measurements.
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 2: ADA assay, bridging format using antibodies HCA210 and HCA211
Figure 2: Ustekinumab was coated on a microtiter plate and left over night; coating concentration for the experiment using product HCA210 as reference standard was 1 µg/ml, and using product HCA211, 1.5 µg/ml. Washing and blocking was performed with 5% BSA in PBST. Anti-ustekinumab antibody, titrated into 10% human serum in the given concentrations, was then added. Detection was performed by adding HRP conjugated ustekinumab* (4 µg/ml in HISPEC asasy diluent), plus QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
*Antibody conjugated to HRP using a LYNX Rapid Conjugated Kit® (product codes LNK001P - LNK006P)
Figure 3: Inhibition of ustekinumab to IL-23 by antibody HCA209
IL-23 (1.0 µg/ml) was used as the coating antigen. A pre-incubated mixture of the anti-ustekinumab antibody, clone AbD18334 (HCA209) was titrated into a constant amount of ustekinumab (0.3 µg/ml).
Free ustekinumab still capable of binding to the plate was detected using HRP conjugated mouse anti-human IgG (Fc) CH2 domain (MCA647P).
The data are shown as the mean of three measurements.
Figure 4: Specificity of anti-ustekinumab antibody HCA211
Figure 4: Demonstration of the specificity of antibodies to ustekinumab. Antigens were coated at 5 µg/ml on a microtiter plate over night. After washing and blocking with 5% milk powder in PBST, HRP conjugated anti-ustekinumab, clone AbD17829_hIgG1 (HCA211), 20µl from a 2µg/ml solution in HISPEC assay diluent, was added. Detection was performed using 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.
STELARA® is a registered trademark of Janssen Biotech, Inc. USA. HuCAL® is a registered trademark of MorphoSys AG. QuantaBlu™ is a trademark of Thermo Fisher Scientific.