(M1330-01-01) Developing a Generic ADA Assay for the Detection of Circulating Immune Complexes Using a Fully Automated Microfluidic Immunoassay System

Purpose: Anti-Drug Antibodies (ADAs) are generated as part of the immune response of a test subject to the introduction of a biological therapeutic. The generation of ADAs can have a deleterious effect on the patients’ health as well as unfavorable impacts on drug efficacy. The detection and measurement of ADAs is a requirement for clinical studies and determining the immunogenic properties of a therapeutic at an early stage can result in better clinical outcomes in the future. Developing multiple specific ADA assays for each drug candidate in preclinical evaluations can be time and resource intensive. By utilizing a generic ADA assay it is possible to screen multiple drug candidates with a single species-specific method. A species-specific generic ADA method for the detection of circulating Drug-ADA immune complexes in mouse serum samples was developed and evaluated. Samples from naïve individuals, as well as those that had been shown to have ADAs present in a drug-specific ADA assay, were used to determine the suitability and performance of this assay.

Methods: ADA analysis in an automated, CD-based format was performed on the Gyrolab microfluidic platform with commercially available Gyrolab Generic ADA kit reagents on mouse serum samples. The positive control used for analysis was a human IgG-mouse IgG dimer. Testing was compared to currently in-use specific methods for the detection of free and complexed ADA. Mouse anti-human ADA:drug immune complexes were detected using a three-step sandwich immunoassay with biotinylated anti-human IgG antibody capture reagent and an Alexa Fluor® 647 labeled anti-mouse IgG monoclonal antibody detection reagent with the Gyrolab Bioaffy 1000 CD.

Results: A dilution series of positive control reagents showed detectable levels of ADA complexes across a 3-log span and specific signal as low as 0.5 ng/mL of positive control. Positive control spiking at 100 ng/mL showed recovery at ≤25% bias in 6 naïve individuals; 1 mouse sample resulted in a bias of 30%. Generic assay reagents used demonstrated inter-run variability at < 2.6% and intra-run variability at 6.9%. The direct comparison with the drug-specific ADA method demonstrates that the generic ADA assay has less unspecific binding and a reduction in the impact of matrix effects, and both the negative control groups and the untreated animals demonstrated no nonspecific binding. One sample, mouse serum test sample 2, showed a large difference between the specific and the generic ADA methods with signals 3-4x higher with the generic assay. Comparison between the methods for detection of free and complexed ADA gave reproducible results with the generic ADA reagents and this generic assay format demonstrated no drug interference.

Conclusion: The automated microfluidic generic ADA assay demonstrated high robustness and reproducibility, demonstrating the suitability of these reagents and platform for immune complex detection in mouse serum.


Three-step sandwich immunoassay with biotinylated anti-human IgG antibody capture reagent and an Alexa Fluor® 647 labeled anti-mouse IgG monoclonal antibody detection reagent


Assay responses for free and complexed ADAs


Standard curve for mouse immune complexes in 10% serum