Proteins are major components of foods, and while the primary interest in proteins is due to their nutritional and functional impact on foods, the allergic reactions induced in sensitized individuals following consumption of certain proteinaceous foods have been a key driver for the development of highly selective and sensitive tools for protein detection and analysis to ensure that food products conform to regulatory standards.
ELISA is a major enzyme-based analytical tool for a broad range of food components and other ingredients, and therefore, an explanation of the underlying principles is appropriate here. Figures 3.1 and 3.2 illustrate the general principles underlying the use of sandwich and competitive ELISA (both of which may be direct or indirect) for protein analysis. In the direct sandwich ELISA, a capture antibody specific for the protein being analyzed is first immobilized onto a solid phase.
When analytes containing the protein of interest are introduced or present in the system or food material, they bind specifically to the immobilized antibody. Detection of such binding is achieved by introducing a second enzyme-labeled analyte-specific antibody into the system, thereby forming a sandwich. In the indirect sandwich ELISA, a second analyte-specific antibody binds to a secondary site on the protein of interest (analyte) to form the sandwich before the enzyme-labeled antibody binding.
The requirement for a secondary binding site or more than one epitope for specific binding makes this type of ELISA primarily applicable to large molecules like proteins. In both types, the binding reaction may be linked to a chromophore.
Competitive ELISA is more flexible and applicable to analysis of small as well as large molecules. In the direct form of the assay, the analyte of interest is first incubated together with the enzyme-labeled antibody and the mixture added to the analyte-specific capture antibody with which they react competitively and in a concentration-dependent manner for detection (Figure 3.2).
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