New Dip and Read Immunogenicity Assay Kit

Sae H. Choo, Director, Assay Development

Introduction

Immunogenicity testing, measuring the immune response to a therapeutic drug, has become an integral part of drug development. The immune system may respond to drug administration by producing anti-drug antibodies (ADA), alternatively called anti-therapeutic antibodies (ATA). Detection of these ADA is essential during the development process, as they could alter the pharmacodynamics (PD) and/or the pharmacokinetics (PK) of the drug.

ADA can be produced in a wide range of concentrations and affinities. To accurately detect all types of these polyclonal antibodies, a biosensing system must have the capacity to work with a wide range of free drug concentrations and matrix types. ForteBio’s Dip and Read Immunogenicity Kit for Octet instruments offers multiple protocols to provide the needed sensitivity, the required drug tolerance, and the flexibility to detect both high- and low-affinity ADA.

ForteBio’s immunogenicity assay protocols avoid extensive washing steps, allowing detection of low affinity antibodies that might be missed using traditional ELISA-based assays. These protocols work across many drug types such as antibodies, proteins, and peptides in both human and animal sample matrix.

The Dip and Read Immunogenicity Kit includes a biosensor that is coated with streptavidin (SA), along with assay reagents. The SA biosensor surface facilitates rapid and stable immobilization of biotinylated drug molecules. ForteBio offers two assay formats, the Enzyme-linked Bridging (BR) assay and a Direct Binding (DB) assay, to provide the flexibility needed to accommodate a wide range of ADA types.

In the BR assay, the SA biosensor is used to rapidly capture the biotin-drug/ADA/fluorescein-drug complex out of the treated sample mixture. By using a standard HRP-linked antibody and precipitating substrate, the Octet instrument provides a rapid, sensitive, and precise readout. With this assay, a 96-well plate can be read in as little as 30 minutes when using the Octet QK384/RED384 system. This protocol often provides enhanced sensitivity compared to the direct binding protocol.

In other cases, the DB assay may be more appropriate, particularly when detecting low-affinity ADA or when the antibody structure does not permit use of a bridging assay. In this assay, the biotin-drug is immobilized onto the SA biosensor surface and the ADA is captured and detected directly in the diluted sample. A 96-well plate can be read in as little as 60 minutes when the biotin-drug immobilization and blocking steps are performed off-line.

Data Analysis

The immunoassays used for screening anti-drug antibodies fall in the class of quasi-quantitative assays due to the lack of similarity between the standard samples and test samples. An upper negative limit of ~95% (1.645 SD) for the cut point (baseline) is recommended.¹ Therefore, determination of assay sensitivity and drug tolerance are based on the statistical clearance from the baseline, not based on the absolute quantification of ADA.

Example Data

Figure 4 shows assay sensitivity and drug tolerance using the BR format. Concentration response curves are shown for sheep anti-human antibody (surrogate ADA) in the presence of varying amount of human IgG, the free drug. The BR format allows sensitivity of 10 ng/mL of free ADA, while tolerating >500 fold in concentration of free drug.

Figure 5 demonstrates assay sensitivity of the DB format in the absence of free drug. Concentration response curves obtained for sheep anti-human antibody (surrogate ADA) show 100 ng/mL sensitivity.

Figure 6 shows assay sensitivity using the DB format in the presence of 10 µg/mL of free drug. Concentration response curves obtained for  sheep anti-human antibody (surrogate ADA) show that the DB assay distinguishes 100 ng/mL of free ADA in the presence of 10 μg/mL of free drug, indicating >100 fold drug tolerance.

Summary and Conclusion

ForteBio’s new Dip and Read Immunogenicity Assay Kit can be used with Octet systems to detect ADAs in both human and animal samples. The assay can be run without plate washing steps in the direct binding format, allowing detection of low affinity antibodies that could be missed in ELISA-type assays. Most importantly, this new kit offers superb sensitivity and drug tolerance to accommodate detection and characterization of this important immune response throughout the drug development process.

REFERENCES

¹ Mire-Sluis AR, Barrett YC, Devanarayan V, Koren E, Liu H, Maia M, Parish T, Scott G, Shankar G, Shores E, Swanson SJ, Taniguchi G, Wierda D, Zuckerman LA. Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J. Imm. Met. 2004; 289(1–2):1–16.

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