Optimization of Serum and Microarray Concentrations for a Semiquantitative Multiplex Indirect Immunoassay

The versatility of protein microarrays provides a variety of possibilities to explore proteomic profiles, allowing researchers to investigate multiple proteins simultaneously with high efficiency. In this study, we employed xMAP® technology to carry out a multiplex indirect semiquantitative immunoassay using the MILLIPLEXⓇ HCYTAAB-17K kit. This kit is designed with an integrated microarray containing 3 positive controls, 1 negative control, and 14 analytes.In this chapter, we provide a detailed protocol for optimizing the serum conditions required to perform the immunoassay. In addition, we have optimized the commercial microarray itself, focusing on reducing non-specific binding and minimizing false positives. Through these improvements, we aim to increase both the sensitivity and specificity of the assay, ensuring more robust and reproducible results in proteomic profiling. The optimization also helps to extend the usability of the kit, making it more versatile for future experiments.