Comparative evaluation of screening approaches for antiviral monoclonal antibody combinations

Monoclonal antibody (mAb) cocktails are increasingly used as antiviral therapeutics, but methods for evaluating mAb combination effects remain poorly standardized. To characterize interactions of recently isolated neutralizing mAbs targeting human cytomegalovirus glycoprotein B, we compared three screening approaches: checkerboard neutralization assays; DiaMOND, a high-throughput small-molecule interaction screening tool modified for mAb combinations; and EMERALD, a modified variable-ratio framework. As the most systematic approach, the checkerboard assay served as the baseline for subsequent comparisons. Most mAb combinations in checkerboard assays were classified as additive or antagonistic according to Loewe additivity-based analyses, regardless of whether the mAbs were competing or non-competing. Apparent synergy was observed primarily at saturating antibody concentrations. When adapted for mAb combinations, the DiaMOND framework produced interaction profiles that differed from checkerboard results under both concentration-based and dose-matched conditions. In contrast, EMERALD captured concentration-dependent interactions across variable mAb ratios and revealed asymmetric behavior, depending on which mAb was held constant. Together, these findings demonstrate that classifications of mAb interactions depend strongly on assay design, concentration range, and analytical framework. They further suggest that robust mAb synergy may be less common than often reported and highlight the need to evaluate antiviral mAb combinations across multiple experimental formats.IMPORTANCEMonoclonal antibody cocktails are gaining foothold as an important strategy for the prevention and treatment of viral infections. Synergistic antibody combinations are often reported, but whether they represent true synergy is unclear because classifications of interactions depend on the experimental design and analytical methods. Moreover, there is no broadly accepted methodology for evaluating interactions among antibody combinations. As a result, it remains unclear whether common approaches for screening antibody combinations accurately capture biologically relevant interactions. Here, we compared four approaches for evaluating antibody combinations to help define best experimental and analytical practices for studying antiviral antibody interactions. Our findings demonstrate that antibody interactions are highly dependent on assay design, choice of antibody concentrations, and timing of antibody exposure, suggesting that synergy and antagonism are not fixed properties of antibody pairs. These findings may be broadly applicable to studies of antiviral antibody combinations across diverse viral systems.