Evaluating Cross-Reactivity in Custom-Made Multiplex Bead-Based Antibody Microarrays: A Methodological Approach

Multiplex antibody microarray assays based on suspension microsphere technology have emerged as a powerful tool in both basic and translational biomedical research, enabling the simultaneous quantification of multiple proteins within a single assay. Developing custom-designed multiplex assays offers the advantage of flexibility beyond predefined biomarker panels; nonetheless, addressing cross-reactivity remains a crucial step to ensure assay reliability and accuracy results. This chapter provides a comprehensive, step-by-step methodology for evaluating cross-reactivity in multiplex antibody microarrays, detailing techniques to validate and optimize assay performance. Additionally, it explores strategies for mitigating cross-reactivity to enhance the precision and interpretability of assay results.