Antibodies for bioanalytical assays to measure alemtuzumab and biosimilar products
Ready-made recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized antibody drug alemtuzumab (MabCampath, LEMTRADA).
These inhibitory (Type 1) antibodies prevent the binding of the drug alemtuzumab to its target CD52, and can therefore be used to detect free drug. They are ideal as capture and detection reagents in pharmacokinetic (PK) bridging assays, and as a surrogate positive control in an anti-drug antibody (ADA) assay.
Catalog Numbers: HCA174, HCA175, HCA175P, HCA198, HCA199
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-idiotypic antibody binding types 1, 2 and 3 and their properties
Table 1. Antibodies Specific to Alemtuzumab
|Catalog #||Clone||Specificity||Formats||Affinity KD, nM||Assay Development Recommendations|
|HCA174||AbD16728||Alemtuzumab||Fab-V5Sx21||1.6||PK bridging ELISA capture antibody|
PK bridging ELISA detection antibody
|2.43||PK bridging ELISA detection antibody with HCA198|
|HCA198||AbD19189||Alemtuzumab||Fab-FH2||0.2||PK bridging ELISA capture antibody with HCA175P|
PK bridging ELISA detection antibody
1 Monovalent Fab antibody, V5-tag and StrepX-StrepX-tag
2 Monovalent Fab antibody, DYKDDDDK-tag& and His-6-tag
3 Affinity measured in the monovalent Fab form
Note: The idiotope of rat anti-CD52 (MCA1642) is identical to Alemtuzumab
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. PK bridging ELISA using antibodies HCA198 and HCA175P.
In figure 1, Anti-Alemtuzumab Antibody clone AbD19189 (HCA198) was coated on a microtiter plate at 1.0 µg/mL and left overnight. After washing and blocking with 5% BSA in PBST, alemtuzumab was spiked in 10% human serum and added in increasing concentrations as indicated. Detection was performed using HRP conjugated Anti-Alemtuzumab Antibody clone AbD16942_hIgG1 (HCA175P) at 4.0 µg/mL in HISPEC Assay Diluent plus QuantaBlu Fluorogenic Peroxidase Substrate.
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 antibody HCA199.
In figure 2, alemtuzumab was coated at 1.5 µg/mL on a microtiter plate overnight. After washing and blocking with 5% BSA in PBST, Anti-Alemtuzumab Antibody clone AbD19189_hIgG1 (HCA199) was added in PBST plus 10% human serum, at the given concentrations. Detection was performed by adding HRP conjugated alemtuzumab, at 4.0 µg/mL in HISPEC Assay Diluent, plus QuantaBlu Fluorogenic Peroxidase Substrate. HRP conjugation of alemtuzumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit.
Fig. 3. Specificity of Human Anti-Alemtuzumab Antibody HCA199.
In figure 3, various antigens were coated at 5.0 µg/mL on a microtiter plate overnight. After washing and blocking with 5% BSA, Human Anti-Alemtuzumab Antibody clone AbD19189_hIgG1 (HCA199) was added at 2.0 µg/mL in HISPEC Assay Diluent. Detection was performed using Mouse Anti-Penta Histidine Tag:HRP Antibody (MCA5995P) plus 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.