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. 2025:2929:97-106.
doi: 10.1007/978-1-0716-4595-6_8.

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

Rocío Paz-González  1 , Patricia Quaranta  1 , Selva Riva-Mendoza  1 , Pablo Domínguez-Guerrero  1 , Francisco J Blanco García  2 , Cristina Ruiz-Romero  3 , Valentina Calamia  3 , Lucía Lourido  3
Affiliations

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

Rocío Paz-González et al. Methods Mol Biol. 2025.

Abstract

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.

Keywords: Antibody microarray; Cross-reactivity; Multiplex microarray; Suspension bead array.

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