Evaluation of Antibody Biotinylation Approaches for Enhanced Sensitivity of Single Molecule Array (Simoa) Immunoassays

In this study, we evaluated the performance of Single Molecule Array (Simoa) immunoassays based on various detection antibody biotinylation approaches. Simoa immunoassays, like other sandwich ELISAs, are highly dependent on the interaction of a biotinylated detection antibody with an enzyme conjugated to streptavidin. Thus, we sought to assess whether different biotinylation reagents can improve the performance and sensitivity of Simoa assays. We selected three proteins, GM-CSF, IFN gamma, and IL-2, that are present at ultralow levels in many biological samples. We compared the performance of these Simoa assays by using five different biotinylation reagents and varying the amount of molar fold excess biotin during the biotinylation process. We found that the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of the Simoa assays, with differences of up to an order of magnitude in sensitivity. We also tested the performance of bulk ELISAs using the different biotinylated detection antibodies and observe differences greater than an order of magnitude in sensitivity. We show that evaluating different strategies for detection antibody biotinylation is a simple approach for optimizing immunoassay performance for enhanced sensitivity.