Previously Unrecognized Nonreproducible Antibody–Probe Interactions

Antibody–antigen (Ab–Ag) interactions are canonically described by a model that exclusively accommodates noninteraction (0) or reproducible interaction (RI) states, yet this model is inadequate to explain often-encountered nonreproducible signals. Here, by monitoring diverse experimental systems using a peptide–protein hybrid microarray, we observed that Ab–probe interactions comprise a substantial proportion of nonreproducible antibody-based results. This enabled our discovery and capacity to reliably identify nonreproducible Ab–probe interactions (NRIs), as well as our development of a powerful explanatory model (“0-NRI-RI-Hook four-state model”) that is mAb concentration-dependent, regardless of specificity, which ultimately shows that both nonspecific interactions and NRIs are not predictable yet certain to happen. Our discoveries challenge the centrality of Ab–Ag interaction specificity data in serology and immunology.