Anti-Tocilizumab Antibodies

Antibodies for bioanalytical method development to measure tocilizumab and biosimilar products

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

Type 1 Anti-Tocilizumab Antibodies

Type 1 Anti-Tocilizumab Antibodies

The Type 1 anti-tocilizumab antibodies specifically recognize the free humanized monoclonal antibody tocilizumab These antibodies do not recognize tocilizumab in complex with soluble or membrane bound human interleukin-6 receptor (IL-6R).

They are ideal for biosimilar development. Suitable for use in direct and 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: HCA252, HCA253, HCA253P, HCA254, HCA255, HCA256

Type 2 Anti-Tocilizumab Antibodies

Type 2 Anti-Tocilizumab Antibodies

The Type 2 anti-tocilizumab antibody does not inhibit the binding of tocilizumab to IL-6R and can therefore detect total drug; free, partially bound and fully bound. This fully human antibody can be used to measure the tocilizumab levels in patient sera.

Product code: HCA257, HCA257P

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 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

Anti-biotherapeutic antibody quality control and characterization


Antibodies Specific to Tocilizumab

Product Code Clone Antibody Specificity Binding Type Format Affinity KD, nM Assay Development Recommendations
HCA252 AbD21362 Tocilizumab Type 1 Fab-FH1 0.9 Indirect ELISA
PK bridging ELISA, capture antibody with HCA253
HCA253 AbD21338_hIgG1 Tocilizumab Type 1 Human IgG1 0.12 Indirect ELISA
PK bridging ELISA, detection antibody with HCA252 or HCA253
ADA assay
HCA253P AbD21338_hIgG1 Tocilizumab Type 1 Human IgG1
HRP labeled
0.12 Direct ELISA
PK bridging ELISA, detection antibody with HCA252 or HCA253
HCA254 AbD21364_hIgG1 Tocilizumab Type 1

Human IgG1

0.32 Indirect ELISA
ADA assay, high affinity
HCA255 AbD21346_hIgG1 Tocilizumab Type 1 Human IgG1 6.22 Indirect ELISA
ADA assay, medium affinity
HCA256 AbD21345_hIgG1 Tocilizumab Type 1 Human IgG1 152 Indirect ELISA
ADA assay, lower affinity
HCA257 AbD22155_hIgG1 Tocilizumab Type 2 Human IgG1 312 Indirect ELISA
PK bridging ELISA, detection antibody with HCA252
HCA257P AbD22155_hIgG1 Tocilizumab Type 2 Human IgG1
HRP labeled
312 Direct ELISA
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


Pharmacokinetic Assay – Bridging ELISA Format

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

 

Figure 1: PK bridging ELISA using antibodies HCA252 and HCA253

Human anti-tocilizumab antibodies, product codes HCA252 and HCA253

Figure 1: Anti-tocilizumab antibody, clone AbD21362 (HCA252) was coated at 1.0 µg/ml on a microtiter plate and left over night. 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 anti-tocilizumab antibody, clone AbD21338 (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: PK bridging ELISA for use with anti-tocilizumab antibodies HCA252 and HCA253


Tocilizumab is inhibited from binding IL-6R by Type 1 neutralizing antibody, but not by Type 2 non-neutralizing antibody

Anti Human IgG HRP Labeled binding

Schematic image of inhibition assay. Drug target capture antigen (red), monoclonal antibody drug (gold), anti-IgG Fc specific detection antibody (gray) labeled with HRP

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

 

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

Human anti-tocilizumab antibodies, product codes HCA252 and HCA257

Figure 2: A microtiter plate was coated over night with soluble human IL-6R at a concentration of 1 µg/ml. After washing and blocking with PBST+5% BSA, a pre-incubated mixture of tocilizumab (0.3 µg/ml) plus increasing concentrations of either human anti-tocilizumab (HCA252) or the monovalent Fab fragment of HCA257 were added. Free tocilizumab, still capable of binding to the human IL-6R coated plate was detected using a mouse anti human IgG (Fc specific) HRP labeled antibody (MCA647P) and QuantaBlu fluorogenic peroxidase substrate. Data are shown as the mean of three measurements. 


Anti-Drug Antibody Assay - Bridging ELISA Format

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

 

Figure 3: ADA bridging ELISA using antibodies HCA253, HCA254, HCA255 and HCA256

Human anti-tocilizumab antibodies, product codes HCA253, HCA254, HCA255 and HCA256

Figure 3: A microtiter plate was coated over night 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 clones HCA253, HCA254, HCA255 and HCA256. Detection was performed using HRP conjugated tocilizumab*. Data are shown as the mean of two measurements.


Determination of Type 2 Antibody Specificity

Determination of Type 2 Antibody Specificity

Figure 4: Specificity of anti-tocilizumab antibody HCA257

Human anti-tocilizumab antibody, product code HCA257P

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 HRP conjugated anti-tocilizumab antibody, clone AbD22155_hIgG1 (HCA257P) at a concentration of 2 μg/ml 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. Actemra® (USA) and RoActemra® (EU) are registered trademarks of Chugai Seiyaku Kabushiki Kaisha Corp., a member of the Roche Group.