Nanoparticle labels have been shown to improve the sensitivity of a sandwich immunoassay significantly. Further improvement in sensitivity is limited by nonspecific binding of the nanoparticle labels. Here, an experimental characterization of assay performance was carried out using clinically important analytes thyroid stimulating hormone and prostate-specific antigen. Particular attention was paid to characterization of nonspecific binding properties of nanoparticle labels. Therefore, different particle sizes and high affinity monoclonal antibodies (Mab) and their Fab and scFv recombinant antibody fragments were investigated. Combination of Fab fragment as a capture antibody and Mab as a detector antibody on a nanoparticle label resulted in high signal-to-background ratio consistently. Against the expectations no significant difference in nonspecific binding was found using fragmented antibodies compared to Mabs. The results also suggested that nonspecific binding was independent of the particle size. The particle size had a significant effect on the specific signal favouring the use of small particles giving a high specific signal. This study indicated that nonspecific binding is not readily affected by the physical size of the nanoparticle label or antibodies used in the assay.