The distribution of inkjet-printed biomolecules in porous nitrocellulose substrates often results in a non-homogeneous spot morphology commonly referred to as 'doughnut-shaped' spots. We have studied the influence of Pluronic F127 (an amphiphilic surfactant) on the functionality of inkjet-printed primary antibody molecules and on the final assay result by performing a one-step antibody binding assay in the nitrocellulose substrate. The primary antibody was printed with and without Pluronic, followed by the addition of double-labelled amplicons as antigen molecules and a fluorophore-labelled streptavidin as detection conjugate. The distribution of the fluorescence intensity down into the nitrocellulose substrate was investigated by confocal laser scanning microscopy in 'Z' stacking mode. Each horizontal slice was further analysed by applying a concentric ring format and the fluorescence intensity in each slice was represented in a colour-coded way. The mean and total fluorescence intensity of the antibody binding assay (fluorescent streptavidin) showed a peak at 0.2% (w/v) Pluronic F127. In addition, an improved spot morphology was observed also peaking at the same Pluronic concentration. Subsequently, we investigated the direct influence of Pluronic F127 on the location of the primary antibody molecules by labelling these molecules with the fluorophore Alexa-488. Our results show that upon increasing the concentration of Pluronic F127 in the printing buffer, the spot diameter increased and the number of primary antibody molecules bound in the spot area gradually decreased. This was confirmed by analysing the distribution of fluorescently labelled primary antibody molecules down into the membrane layers. We conclude that a particular ratio between primary antibody and Pluronic F127 molecules in combination with available substrate binding capacity results in an optimal orientation, that is Fab-UP, of the primary antibody molecules. Consequently, an increased number of antigen molecules (in our case the labelled amplicons) and of the fluorescent detection conjugate (streptavidin) will give an optimal signal. Moreover, distribution of the primary antibody molecules was more homogeneous at the optimal Pluronic F127 concentration, contributing to the better spot morphology observed.