LAUSR

Antibody microarrays enable multiplexed protein detection with minimal reagent consumption, but they continue to be plagued by lack of reproducibility. Chemically functionalized glass slides are used as substrates, yet antibody binding spatial inhomogeneity across the slide has not been analyzed in antibody microarrays. Here, we characterize spatial bias across five commercial slides patterned with nine overlapping dense arrays (by combining three buffers and three different antibodies), and we measure signal variation for both antibody immobilization and the assay signal, generating 270 heatmaps. Spatial bias varied across models, and the coefficient of variation ranged from 4.6 to 50%, which was unexpectedly large. Next, we evaluated three layouts of spot replicates-local, random, and structured random-for their capacity to predict assay variation. Local replicates are widely used but systematically underestimate the whole-slide variation by up to seven times; structured random replicates gave the most accurate estimation. Our results highlight the risk and consequences of using local replicates: the underappreciation of spatial bias as a source of variability, poor assay reproducibility, and possible overconfidence in assay results. We recommend the detailed characterization of spatial bias for antibody microarrays and the description and use of distributed positive replicates for research and clinical applications.