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.抗体-抗原（Antibody–antigen, Ab–Ag）相互作用的经典模型仅能适配无相互作用（0）或可重复相互作用（Reproducible Interaction, RI）两种状态，但该模型无法解释实验中频繁出现的不可重复信号。本研究通过肽-蛋白杂交微阵列监测多种实验体系，发现抗体-探针相互作用在基于抗体的实验结果中占不可重复结果的相当比例。基于该发现，我们得以可靠识别不可重复抗体-探针相互作用（Nonreproducible Ab–probe Interactions, NRIs），并开发出一款普适性强的解释模型——“0-NRI-RI-Hook四态模型”，该模型不受抗体特异性影响，仅依赖单克隆抗体浓度，最终证实非特异性相互作用与NRIs均无法预测，但必然会发生。本研究的发现挑战了抗体-抗原相互作用特异性数据在血清学与免疫学领域的核心地位。