Consistent performance of anti-drug antibody (ADA) assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS) buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB) had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC) serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.