The simultaneous detection of multidomain ADAs using a multiplex assay optional subtitle: a multiplexed approach to multidomain ADA detection

The increasing prevalence of bispecific and multidomain therapeutics has introduced significant challenges for immunogenicity assessment, as conventional approaches rely on separate anti-drug antibody (ADA) assays for each therapeutic domain. This paradigm increases sample consumption, extends development timelines, adds operational complexity, and complicates cross-domain data interpretation.

Here, we describe the development and prevalidation performance of a multiplexed, domain-specific immunogenicity assay based on Luminex^Ⓡ xMAP^Ⓡ technology. Pegylated Cas9 was selected as a multidomain model construct because it combines a highly immunogenic protein domain with a polymer moiety known to elicit immune responses in human populations, providing a clinically relevant test case for multiplex ADA detection.

The assay enables simultaneous detection of ADAs directed against multiple domains within a single workflow. Key features include on-demand biotin-avidin bead coupling, adjustable bead ratios to accommodate differing minimum required dilutions (MRDs), and assay sensitivity consistent with regulatory expectations.

Collectively, these results demonstrate the feasibility of a unified multiplexed platform for clinical immunogenicity assessment of multidomain therapeutics, enabling domain-specific ADA characterization while reducing assay burden and sample requirements.