Initiator Integrated Poly(dimethysiloxane)-Based Microarray as a Tool for Revealing the Relationship between Nonspecific Interactions and Irreproducibility

Abstract

Nonspecific interactions (NSIs) and irreproducibility greatly reduce the accuracy of antigen–antibody screening, which is key to the discovery of monoclonal antibody drugs and biomarkers identification. We previously developed a solid supporting material, polymer-coated initiator integrated poly­(dimethysiloxane) (iPDMS), which is able to provide near-zero background for microarray screening. Here, we applied two monoclonal antibodies (mAbs), namely, anti-FLAG and HM1, to screen an iPDMS-based peptide microarray with 2083 peptides from 62 proteins to evaluate NSIs and irreproducibility. In addition to recognizing their cognate epitopes, the two mAbs also cross-reacted with random sequences, especially when they were used at high concentrations. At 50 μg mL^–1, 295 peptides (14.2% of the peptide library) had positive reactions to anti-FLAG and only 39 peptides (1.9%) reacted positively to HM1. Virtually all cross-reactions disappeared when the [mAbs] reached 0.01 μg mL^–1. Reproducible experiments of 404 peptides at various [mAbs] showed that only specific interactions, molecular mimicry, and mimotope were reproducible between different experiments. These findings suggest that irreproducibility was at least partially caused by NSIs. We also demonstrated that repeating tests and mAb dilution could effectively avoid NSI-related irreproducibility in serological screening. This will not only largely simplify the data analysis, but will also make immunoassays more reliable for clinical research