ADA Assay Development on MSD: Practical Execution and Critical Concepts
Introduction
Anti-drug antibody (ADA) assays are critical for evaluating drug safety and are required throughout pre‑clinical and clinical development. In a standard bridging ADA assay, anti-drug antibodies in the sample “link” a capture reagent and a detection reagent, forming a “bridge” that generates a measurable signal. While this format may seem straightforward in theory, translating it into a reliable, fit-for-purpose method requires a series of deliberate design decisions. FDA guidance instructs that samples should be evaluated in a tiered sequence: screening to flag potential positives; confirmatory testing to verify drug-specific responses; and titer to characterize their magnitude (FDA, 2019). Each of these staged tests introduces unique design considerations that ultimately influence the assay’s sensitivity, specificity, robustness, and overall performance under real study conditions.
Because these design decisions depend on how the assay generates and measures signal, the choice of detection platform becomes an integral part of method development. The MSD platform is commonly used for bridging ADA assays because its electrochemiluminescent detection technology provides strong signal to low background performance and a broad analytical range, making it well suited for reliable detection of low-level ADA responses.
The following overview highlights key considerations in ADA assay development using the MSD instrument, including assay design, reagent preparation, cut-point determination, and other factors affecting assay performance. At Emery Pharma, these activities are approached with an emphasis on understanding how each decision contributes to the generation of meaningful, reproducible data.
What “Fit-for-Purpose” Really Means
A fit-for-purpose ADA assay is one that is appropriately sensitive, precise, and robust for its intended use. In practice, this means the assay must be capable of detecting true, specific anti-drug antibodies while maintaining consistency across runs and sample types. Achieving this requires balancing multiple performance characteristics. For example, prioritizing sensitivity can inadvertently sacrifice screening specificity, yielding an excessive number of false positives. A well-designed method is optimized so that sensitivity, precision, and specificity work together, ensuring the screening tier is sensitive enough to catch true positives, while the confirmatory tier accurately isolates drug-specific responses.
The Bridging Format: How Detection Works
Bridging assays are designed around the ability of anti-drug antibodies to bind two drug molecules at once. This has the advantage of specific ADA directed antibodies that can provide a lower background.
Figure 1. Bridging ADA Assay Format.
By labeling the drug with complementary tags, the assay enables the anti-drug antibodies to form a bridge that can be measured as a signal related to specific ADA binding. This format is widely used because it supports sensitive detection, but its effectiveness depends on how well these interactions are formed and detected during assay development. On the MSD platform, electrochemiluminescence detection provides a sensitive means of measuring these bridging interactions, supporting reliable ADA detection when assay conditions have been appropriately optimized. This approach reduces the background noise, producing a cleaner result.
Defining Assay Performance: The Positive Control
Positive controls serve as a reference point for how the assay behaves when ADA is present. While the choice between polyclonal and monoclonal antibodies involves real tradeoffs, an appropriate control is ultimately defined by how it performs in the assay. Controls are typically run at multiple concentrations to ensure that the entire range is providing reproducible detection. A low control near the assay’s sensitivity limit confirms detection of weak responses, and higher controls monitor performance across the range. Together, these considerations make the control a reliable benchmark for tracking assay behavior over time.
Reagent Preparation: Why Conjugation Matters
The process of attaching tags such as biotin and ruthenium to the drug is a critical and often underappreciated step. These labeled reagents serve distinct roles in the assay: the biotin tag enables capture of the complex on the plate, while the ruthenium tag generates the measurable signal. The ruthenium label is the electrochemiluminescent tag used for detection on the MSD platform, available as MSD’s SULFO-TAG labeling reagent.
Because signal generation on MSD depends on the interaction between the SULFO-TAG label and the instrument’s electrochemical stimulation, optimization of conjugation conditions can have a significant impact on assay performance. Careful characterization of labeled reagents with well-defined labeling reactions helps maximize signal quality while maintaining the binding characteristics required for effective ADA detection. These considerations make conjugate optimization a key component of ADA assay development on the MSD platform.
The quality and reproducibility of the conjugates directly affects both signal strength and consistency. During assay development, different conjugation conditions are evaluated to find the right balance. Heavily labeled conjugates can generate increased background due to lack of specificity, or altered binding, while insufficient labeling can reduce sensitivity. For this reason, conjugates are evaluated experimentally rather than assuming a particular labeling approach will perform optimally and reproducibly.
Facilitating Detection in Real Samples: Acid Pretreatment
In samples where drug is present, anti-drug antibodies may already be bound in complexes that prevent detection. Acid pretreatment is used to temporarily disrupt these complexes, allowing the anti-drug antibodies to be measured. This step improves the assay’s ability to detect ADA even when drug is present in the sample. This property is known as drug tolerance, and it is critical for accurate assessment in clinical studies. ADA measurement is often performed at decreased drug concentrations, but since this may not be possible with some dosing regiments it is worthwhile to consider measurement of ADA while in the presence of drug.
Acid pretreatment is a standard bioanalytical approach in ADA. That being said, acid pretreatment can affect protein stability. It is recommended to assess anti-drug antibodies before and after acid pretreatment.
Managing Biological Variability: Matrix Considerations
Biological samples can also introduce variability that can affect assay performance. Differences between individual donors and other matrix components can produce nonspecific background and influence signal behavior. To mitigate this possible effect, samples are diluted to a minimum required dilution (MRD), the smallest dilution that reduces this background to an acceptable level while preserving the sensitivity needed to detect ADA. Evaluating and accounting for these matrix effects helps ensure that the assay performs reliably across diverse sample sets.
Setting Meaningful Thresholds: Cut-Point Determination
The cut-point is the signal threshold that distinguishes potentially positive samples from negative ones. Rather than relying on arbitrary or predefined values, ADA assays establish it statistically, based on variability observed in drug-naive samples. By analyzing a minimum of 50 drug-naive donors, a cut-point can be set that balances sensitivity with an acceptable false-positive rate. This ensures that results are interpreted consistently and objectively. This is a critical part of the ADA tiered strategy as all three parts of the assay need to have their own cut-point.
The Tiered Strategy: Building Confidence in Results
ADA testing typically follows a tiered approach consisting of screening, confirmatory, and titer assays. Together, these stages ensure that potential ADA-positive samples are identified, verified, and evaluated for the relative level of anti-drug antibodies present.
Figure 2. Tiered testing strategy: screening, confirmatory, and titer assays.
Screening Assay
The screening assay is designed to identify samples that may contain anti-drug antibodies. To maximize sensitivity, the screening cut-point is established statistically using drug-naive samples and is typically set at the 95th percentile, corresponding to a 5 percent false-positive rate. This approach helps minimize the risk of failing to identify samples that truly contain ADA, while recognizing that some samples without ADA may also be flagged for further evaluation.
Confirmatory Assay
Because screening assays intentionally accept a limited false-positive rate, samples that screen positive must undergo confirmatory testing. In a confirmatory assay, excess drug is added to the sample, with the amount established during assay development. If the observed signal is due to ADA binding the drug, the added drug competes for binding and reduces the assay signal. This competitive inhibition demonstrates that the response is drug-specific rather than the result of nonspecific interactions.
Titer Assay
Samples confirmed to contain ADA undergo titer assessment to characterize the level of immune response. Titer is typically determined through serial dilution of the sample and identification of the highest dilution that remains positive relative to the titer cut-point. Unlike quantitative ligand-binding assays that report absolute concentrations, titer is a comparative value useful for tracking how a response changes across samples or over time.
A Program-Level Perspective
Effective ADA assay development on the MSD platform requires more than executing individual steps. At Emery Pharma, it involves understanding how each decision contributes to the overall performance of the assay within a study. Taking a holistic approach ensures that the method is not only technically sound but also aligned with the broader goals of the program. Ultimately, successful ADA development on the MSD platform is the result of a series of deliberate decisions that collectively contribute to the generation of reliable and reproducible data.
At Emery Pharma we perform MSD ADA development, validation and clinical sample testing in a detailed and regulatory compliant manner. Contact us to discuss how we can help you with your ADA study!
References
FDA. 2019. Immunogenicity Testing of Therapeutic Protein Products—Developing and Validating Assays for Anti-Drug Antibody Detection. Guidance for Industry. U.S. Food and Drug Administration.
Originally authored by Zachary Martinez. This article was reviewed by Dr. Janet Liu, current Director of Biology.
