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Ready-made recombinant, anti-idiotypic monoclonal antibodies, highly specific for the human antibody drug adalimumab (HUMIRA).
The Type 1 anti-adalimumab antibodies inhibit the binding of the drug adalimumab to its target, TNFα, and therefore detect free drug. These antibodies are ideal for use in direct or indirect 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.
Product codes: HCA202, HCA203, HCA204, HCA204P, HCA205
The Type 3 antibodies specifically recognize the adalimumab/TNFα complex. These antibodies detect adalimumab only when it is bound to TNFα. They do not bind to free adalimumab or TNFα. With these antibodies, PK assays can be built that avoid the bridging format. In addition, they allow detection of adalimumab bound to its target in serum as opposed to free adalimumab.
Product codes: HCA206, HCA207, HCA231, HCA232, HCA232P
This fully human antibody is derived from the same parent clone as HCA204 and has been converted to an IgE isotype. Most ADAs in patient serum have isotype IgG, but ADAs with isotype IgE can potentially elicit anaphylactic reactions. This IgE isotype antibody has been developed for use as a reference standard in an ELISA to measure IgE ADAs in patient samples.
Product code: HCA271
The recombinant anti-adalimumab 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 perfect 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|
Direct and indirect ELISA
PK bridging ELISA with HCA204
|HCA203||AbD18654_hIgG1||Adalimumab||Type 1||Human IgG1||0.15*||
|HCA204||AbD18655_hIgG1||Adalimumab||Type 1||Human IgG1||0.06*||
PK bridging ELISA with HCA202
PK bridging ELISA with HCA202
|HCA271||AbD18655_hIgE||Adalimumab||Type 1||Human IgE||0.06*||ADA assay for IgE|
|HCA205||AbD18664_hIgG1||Adalimumab||Type 1||Human IgG1||10.5*||
|HCA206||AbD18754||Adalimumab/TNFα||Type 3||Fab-FH1||67||PK antigen capture format|
|HCA207||AbD18754_hIgG1||Adalimumab/TNFα||Type 3||Human IgG1||67*||PK antigen capture format|
|HCA2312||AbD20350||Adalimumab/TNFα||Type 3||Fab-FH1||3.2||PK antigen capture format|
|HCA2322||AbD20349_hIgG1||Adalimumab/TNFα||Type 3||Human IgG1||6.7*||PK antigen capture format|
|6.7*||PK antigen capture format|
Table 1 Antibody Specifications
2HCA231 and HCA232/HCA232P are affinity matured versions of clone 18754 (HCA206 and HCA207, respectively)
*Affinity measured in the monovalent Fab form
Adalimumab drug target Recombinant human TNF alpha (PHP051)
Adalimumab drug isotype control Recombinant human IgG1 kappa (HCA192)
Adalimumab biosimilar for research use Human Anti-TNF alpha Antibody (MCA6088)
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: Adalimumab PK ELISA bridging format using antibodies HCA202 and HCA204P
Figure 1: Anti-adalimumab antibody, clone AbD18654 (HCA202) was coated on a microtiter plate at 1.0 µg/ml and left over night. Washing and blocking was performed with 5% BSA in PBST. Adalimumab titrated into 10% human serum was added. Detection was performed using HRP conjugated anti-adalimumab antibody clone AbD18655_hIgG1 (HCA204P) diluted to 2 µg/ml in HISPEC assay diluent (BUF049), plus QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
Schematic image of PK antigen capture format ELISA. Drug target (red), monoclonal antibody drug (gold), drug target complex detection antibody, Fab format (purple), labeled with HRP
Figure 2: Adalimumab PK ELISA antigen capture format using antibody HCA231
Figure 2: Recombinant human TNFα (PHP051) was coated on a microtiter plate at 5 µg/ml. After washing and blocking with 5% BSA in PBST, adalimumab spiked into 10% human serum was added in the given concentrations. After washing, either unlabeled or HRP labeled anti-adalimumab/TNFα complex specific antibody, clone AbD20350 (HCA231) was added at 2µg/ml in PBST. Unlabeled HCA231 was detected by adding anti-Penta Histidine Tag:HRP (MCA5995P) in HISPEC assay diluent (BUF049) and QuantaBlu fluorogenic peroxidase substrate. HRP-labeled HCA231 was detected directly with QuantaBlu substrate. Data are shown as the mean of three measurements. Antibodies were conjugated to HRP using a LYNX Rapid Conjugation Kit® (LNK001P-LNK006P).
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 assay, bridging format using antibodies HCA203, HCA204 and HCA205
Figure 3: Adalimumab was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, the anti-adalimumab antibodies in human IgG1 format (black squares adalimumab antibody clone AbD18654_hIgG1 (HCA203); red circles adalimumab antibody clone AbD18655_hIgG1 (HCA204); green triangles adalimumab antibody clone AbD18664_hIgG1 (HCA205) spiked in 10% human serum in the given concentrations were added. Detection was performed by adding HRP conjugated adalimumab, 2 µg/ml in HISPEC assay diluent (BUF049), plus QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements.
Protocol: ADA bridging ELISA protocol adalimumab
Schematic image of ADA bridging assay for IgE isotype detection. Monoclonal antibody drug as capture antibody (gold), fully human anti-idiotypic antibody, IgE format (blue), detection with HRP conjugated anti-human IgE antibody (gray).
Figure 4: IgE isotype ADA assay, using antibodies HCA271 and 0100-0413P
Figure 4: A microtiter plate was coated over night with adalimumab, human IgG1 kappa or human IgG1 lambda control at a concentration of 5 μg/ml. After washing and blocking with PBST+5% BSA, detection was performed using human anti-adalimumab antibody clone AbD18655_hIgE (HCA271), titrated to the given concentrations in PBST+10% NHS, followed by HRP conjugated mouse anti-human IgE (0100-0413P) in HISPEC assay diluent (BUF049) and QuantaBlu fluorogenic peroxidase substrate. Data for binding to adalimumab and the controls are shown as the mean of three measurements.
Figure 5: Inhibition of adalimumab binding to TNFα using antibodies HCA203 and HCA207
Figure 5: Recombinant human TNFα (PHP051) was coated on a microtiter plate at 1.0 µg/ml, followed by a pre-incubated mixture of the anti-adalimumab antibody, clone AbD18654_hIgG1 (HCA203) titrated into a constant amount of adalimumab (0.3 µg/ml). Free adalimumab, still capable of binding to the plate, was detected using HRP conjugated anti-adalimumab/TNFα complex antibody, clone AbD18754_hIgG1 (HCA207). Data are shown as the mean of three measurements.
Figure 6: Demonstration of the specificity of antibody HCA232 to the adalimumab/TNFα complex.
Figure 6: Type 3 antibodies (HCA206, HCA207, HCA231 and HCA232) will detect adalimumab only when bound to its target TNFα. 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 HRP-conjugated anti-adalimumab/TNFα complex-specific antibody, clone AbD20349_hIgG1 (HCA232P) and QuantaBlu fluorogenic peroxidase substrate.
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