Anti-Vedolizumab Antibodies

Antibodies for bioanalytical and patient monitoring assays for vedolizumab and biosimilar products

Recombinant monoclonal anti-idiotypic antibodies, highly specific for the humanized IgG1 kappa antibody drug vedolizumab (Entyvio)

 Anti-Vedolizumab Inhibitory Antibodies

Anti-Vedolizumab Inhibitory Antibodies

These antibodies inhibit the binding of the drug vedolizumab to its target, α4β7 integrin, and therefore detect free drug. A pair of these antibodies is ideal for the development of a pharmacokinetic (PK) bridging ELISA. Three antibodies of high, medium or low affinity are suitable as a reference standard for an anti-drug antibody (ADA) assay.

 

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-biotherapeutic antibodies and their binding types and properties

Anti-biotherapeutic antibody quality control and characterization

Table 1. Antibodies Specific to Vedolizumab

Catalog #

Clone

Antibody Specificity

Binding Type

Format

Affinity
KD, nM

Assay Development Recommendations

HCA292

AbD30136

Vedolizumab

Inhibitory

Fab-FH1

0.1

PK bridging ELISA capture antibody

HCA293

AbD30136_hIgG1

Vedolizumab

Inhibitory

Human IgG1

0.12

PK bridging ELISA detection antibody

ADA assay

HCA294

AbD30138_hIgG1

Vedolizumab

Inhibitory

Human IgG1

1.52

PK bridging ELISA detection antibody

ADA assay

HCA294P

AbD30138_hIgG1

Vedolizumab

Inhibitory

Human IgG1
HRP labeled

1.52

PK bridging ELISA detection antibody

HCA295

AbD30139_hIgG1

Vedolizumab

Inhibitory

Human IgG1

42

PK bridging ELISA detection antibody

ADA assay

1 F=DYKDDDDK-tag H=His-6-tag
2 Affinity measured in the monovalent Fab format

Related Product

Vedolizumab drug isotype control Recombinant Human IgG1 Kappa (HCA192)


Pharmacokinetic Assay

Schematic image of PK Bridging ELISA

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. Vedolizumab PK ELISA bridging format using antibodies HCA292 and HCA294P.

Fig. 1. Vedolizumab PK ELISA bridging format using antibodies HCA292 and HCA294P.

Measurement of free drug with Human Anti-Vedolizumab Antibodies HCA292 and HCA294P

In Figure 1, Human Anti-Vedolizumab Antibody, clone AbD30136 (HCA292) was coated on a microtiter plate at 1 µg/ml and left overnight. Washing and blocking was performed with PBST + 5% BSA. PBST with 10% human serum, and spiked with increasing concentrations of vedolizumab was added. Detection was performed using HRP conjugated Human Anti-Vedolizumab Antibody, clone AbD30138_hIgG1 (HCA294P) diluted to 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 protocol vedolizumab HCA292 and HCA294P 


Anti-Drug Antibody Assay – Bridging ELISA 

Schematic image of ADA bridging assay.

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 antibodies HCA293, HCA294 and HCA295.

Fig. 2. ADA bridging ELISA using antibodies HCA293, HCA294 and HCA295.

Immunogenicity assay using Human Anti-vedolizumab Antibodies as calibration standard

In Figure 2, vedolizumab was coated at 1 µg/ml on a microtiter plate overnight. After washing and blocking with PBST + 5% BSA, PBST with 10% human serum was added, spiked with increasing concentrations of Human Anti-Vedolizumab Antibody, clones AbD30136_hIgG1 (HCA293), AbD30138_hIgG1 (HCA294) or AbD30139_hIgG1 (HCA295). Detection was performed using HRP conjugated vedolizumab at 2 µg/ml in HISPEC Assay Diluent, and QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements. HRP conjugation of vedolizumab was performed using a LYNX Rapid HRP Antibody Conjugation Kit®.

Protocol: ADA bridging ELISA protocol vedolizumab HCA293, HCA294, HCA295


Inhibition ELISA

Fig. 3. Demonstration of the inhibitory property of antibody HCA292

Fig. 3. Demonstration of the inhibitory property of antibody HCA292.


In Figure 3, recombinant human α4β7 integrin was coated on a microtiter plate at 1 µg/ml overnight. After washing and blocking with PBST + 5% BSA, vedolizumab was added (0.3 µg/ml), spiked with increasing concentrations of Human Anti-Vedolizumab Antibody HCA292. Free vedolizumab, still capable of binding to the integrin coated plate, was detected using Mouse Anti-Human IgG (Fc) CH2 Domain:HRP Antibody (MCA647P), followed by QuantaBlu Fluorogenic Peroxidase Substrate. Data are shown as the mean of three measurements.


Demonstration of Antibody Specificity

Specificity of Human Anti-Vedolizumab Antibody HCA295
Fig.4. Specificity of Human Anti-Vedolizumab Antibody HCA295.

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-Vedolizumab Antibody, clone AbD30139_hIgG1 (HCA295) 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.