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Enzyme-Based Immunoassay Techniques

Book Title: Molecular Diagnostics and Genomic Medicine: Transforming Healthcare with AI

Editors: Dr. Alok Kumar Srivastav, Dr. Priyanka Das, Dr. Babasaheb Ghodke, and Dr. Nikita Pathak

Chapter: 7

DOI: https://doi.org/10.59646/978-81-69297-97-4_7

Authors: Babasaheb Ghodke, Priyanka Das, Alok Kumar Srivastav, Nikita Pathak

Abstract

Enzyme immunoassays (EIAs) have become essential tools in modern diagnostics by combining antibody-antigen specificity with enzymatic signal amplification for highly sensitive analyte detection. The chapter examines key enzyme systems such as horseradish peroxidase and alkaline phosphatase, emphasizing their selection based on stability, activity, and detection compatibility. It explores conjugation techniques, including glutaraldehyde crosslinking and biotin-streptavidin systems, which ensure effective enzyme labeling and signal generation. Solid-phase platforms, particularly microplate and membrane-based systems, are discussed alongside detection formats such as colorimetric, chemiluminescent, and fluorescent methods. The distinction between homogeneous and heterogeneous immunoassays highlights differences in workflow, sensitivity, and clinical applications. Advanced applications in Western blotting and immunohistochemistry demonstrate protein detection and localization capabilities. The role of monoclonal and polyclonal antibodies is analyzed in optimizing assay performance. Collectively, EIAs enable rapid, accurate diagnostics, particularly in clinical microbiology and disease detection.

Keywords: Enzyme immunoassay, ELISA, Antibody-antigen interaction, Detection systems, Immunodiagnostics, Clinical microbiology.

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