Spatial Bias in Antibody Microarrays May Be an Underappreciated Source of Variability

Abstract

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.

Compartmentalized Linker Array: A Scalable and Transferrable Microarray Format for Multiplexed Immunoassays

“…In this method, the multiple micro-spots in each compartment function as the technical replicates for immunoassays. The signals of the spots on the edge of each compartment near the hydrophobic barriers could be affected by the edge effect during the bioreagent incubation , and the autofluorescence of the hydrophobic barriers. In each 2 mm × 2 mm compartment, we tested the spot layout of four (2 × 2 array) and nine (3 × 3 array) spots, with the diameter of each spot approximately 300 μm and the spot center-to-center distance approximately 700 μm.…”

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

“…The slight autofluorescence of the aldehyde coating produced a visible signal, and slides with immediately visible irregularities were discarded. The possible impact of spatial bias on EV microarrays assay reproducibility and variability was not studied in depth, however …”

“…Reagent-driven cross-reactivity can arise when mixing multiple detection antibodies 47 and lead to false positive signals, such as the ones observed here with our negative anti-GFP control in some experiments. We previously identified spatial bias as a likely underestimated source of variability in antibody microarrays, 63 and it is of particular concern when multiple samples are measured at different locations on a slide. We performed a visual, qualitative check of each functionalized slide prior to the assay by scanning pristine slides with strong signal gain in the 532 nm channel.…”

Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

“…Reagent-driven cross-reactivity can arise when mixing multiple detection antibodies 62 and lead to false positive signals, such as the ones observed here with our negative anti-GFP control in some experiments. We previously identified spatial bias as a likely underestimated source of variability in antibody microarrays, 63 and it is of particular concern when multiple samples are measured at different locations on a slide. We performed a visual, qualitative check of each functionalized slide prior to the assay by scanning pristine slides with strong signal gain in the 532 nm channel.…”

“…The possible impact of spatial bias on EV microarrays assay reproducibility and variability was not studied in depth, however. 63 The addition of oligonucleotide barcoding circumvents common multiplexing issues such as species cross-reactivity and limited availability of species or directly labeled antibodies. The best suited primary antibody can be used for every target, independently of its species, clonality, or availability as a conjugate.…”