Anti-Tocilizumab Antibodies

Antibodies for bioanalytical method development to measure tocilizumab and biosimilar products

Ready-made recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized antibody drug tocilizumab (Actemra/RoActemra).

Type 1 Anti-Tocilizumab Antibodies

Anti-Tocilizumab Inhibitory Antibodies

These inhibitory (Type 1) antibodies specifically recognize the free humanized monoclonal antibody tocilizumab. They do not recognize tocilizumab in complex with soluble or membrane bound human interleukin-6 receptor (IL-6R). An antibody pair is ideal for the development of a pharmacokinetic (PK) bridging ELISA to detect free drug. Four antibodies of different affinities are suitable as a surrogate positive control for an anti-drug antibody (ADA) assay.

Catalog Numbers: HCA252, HCA253, HCA253P, HCA254, HCA255, HCA256

Type 2 Anti-Tocilizumab Antibodies

Anti-Tocilizumab Non-Inhibitory Antibody

The non-inhibitory (Type 2) antibody does not inhibit the binding of tocilizumab to IL-6R and can therefore detect drug bound to its target. It can be used as a detection antibody in a PK bridging assay to measure free and partially bound drug.

Catalog Numbers: HCA257, HCA257P

These 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. Antibodies are fully human, so they are also ideal as controls or calibrators for ADA assays. The in vitro production of recombinant antibodies results in a consistent, secure supply of these critical reagents for use throughout preclinical development and clinical trials.

More information about anti-idiotypic antibody binding types 1, 2 and 3 and their properties

Anti-biotherapeutic antibody quality control and characterization


Table 1. Antibodies Specific to Tocilizumab

Catalog # Clone Antibody Specificity Binding Type Format Affinity KD, nM Assay Development Recommendations
HCA252 AbD21362 Tocilizumab Inhibitory Fab-FH1 0.9 PK bridging ELISA
capture antibody with HCA253
HCA253 AbD21338_hIgG1 Tocilizumab Inhibitory Human IgG1 0.12 PK bridging ELISA
detection antibody with HCA252 or HCA253
ADA assay
HCA253P AbD21338_hIgG1 Tocilizumab Inhibitory Human IgG1
HRP labeled
0.12 PK bridging ELISA
detection antibody with HCA252 or HCA253
HCA254 AbD21364_hIgG1 Tocilizumab Inhibitory Human IgG1 0.32 ADA assay, high affinity
HCA255 AbD21346_hIgG1 Tocilizumab Inhibitory Human IgG1 6.22 ADA assay, medium affinity
HCA256 AbD21345_hIgG1 Tocilizumab Inhibitory Human IgG1 152 ADA assay, lower affinity
HCA257 AbD22155_hIgG1 Tocilizumab Non-inhibitory Human IgG1 312 PK bridging ELISA
detection antibody with HCA252
HCA257P AbD22155_hIgG1 Tocilizumab Non-inhibitory Human IgG1
HRP labeled
312 PK bridging ELISA
detection antibody with HCA252

Table 1 Antibody Specifications
1 F=DYKDDDDK-tag; H=His-6-tag
2 Affinity measured in the monovalent Fab form

Related Products

Tocilizumab drug isotype control Recombinant Human IgG1 kappa (HCA192)
Tocilizumab biosimilar for research use Human Anti-IL6R Antibody (MCA6106)


Pharmacokinetic Assay

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 bridging ELISA using antibodies HCA252 and HCA253

 

Fig. 1. PK bridging ELISA using antibodies HCA252 and HCA253P.

Measurement of free drug with human anti-tocilizumab antibodies

In figure 1, Human Anti-Tocilizumab Antibody, clone AbD21362 (HCA252) was coated at 1.0 µg/ml on a microtiter plate and left overnight. After washing and blocking with PBST + 5% BSA, 10% human serum was added spiked with increasing concentrations of tocilizumab. Detection was performed by adding HRP conjugated Human Anti-Tocilizumab Antibody, clone AbD21338_hIgG1 (HCA253P) 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.

Protocol: Tocilizumab PK bridging ELISA protocol HCA252 and HCA253P


Inhibition ELISA

Inhibition of tocilizumab binding to human IL-6R by HCA252, but not by HCA257

 

Fig. 2. Inhibition of tocilizumab binding to human IL-6R by HCA252, but not by HCA257. 

In figure 2, a microtiter plate was coated overnight with soluble human IL-6R at a concentration of 1 µg/ml. After washing and blocking with PBST + 5% BSA, tocilizumab was added (0.3 µg/ml), spiked with increasing concentrations of inhibitory Human Anti-Tocilizumab Antibody, clone AbD21362  (HCA252), or non-inhibitory Human Anti-Tocilizumab Antibody, clone AbD22155 (HCA257), in the monovalent Fab format. Free tocilizumab, still capable of binding to the human IL-6R coated plate was detected using a Mouse Anti-Human IgG (Fc) CH2 Domain:HRP Antibody (MCA647P) and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. 


Anti-Drug Antibody Assay - Bridging ELISA

Four anti-tocilizumab 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)

ADA bridging ELISA using antibodies HCA253, HCA254, HCA255 and HCA256

 

Fig. 3. ADA bridging ELISA using antibodies HCA253, HCA254, HCA255 and HCA256

Immunogenicity assay using human anti-tocilizumab antibodies

In figure 3, a microtiter plate was coated overnight with tocilizumab 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 the different human anti-tocilizumab antibodies HCA253, HCA254, HCA255 or HCA256. Detection was performed using HRP conjugated tocilizumab. Data are shown as the mean of two measurements. HRP conjugation of tocilizumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit.


Demonstration of Type 2 Antibody Specificity

Determination of Type 2 Antibody Specificity

Fig. 4. Specificity of Non-Inhibitory Anti-Tocilizumab Antibody HCA257.

In figure 4, a microtiter plate was coated overnight with various antigens at a concentration of 5 μg/ml. After washing and blocking with PBST + 5% BSA, detection was performed using HRP conjugated Human Anti-Tocilizumab Antibody, clone AbD22155_hIgG1 (HCA257P) at a concentration of 2 μg/ml in HISPEC Assay Diluent (BUF049A) and 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.