Subsequently, the antigen is allowed to react with a specific antibody, which is detected by an enzyme-labeled secondary antibody. The antigen in the fluid phase is immobilized on a solid phase, such as a microtiter plate constituting rigid polystyrene, polyvinyl chloride, and polypropylene.
To detect these molecules, an antigen or antibody is labeled using enzymes, the so-called enzyme immunoassay, in which alkaline phosphatase (ALP), horseradish peroxidase (HRP), and β-galactosidase are commonly used. Hence, by exploiting this reaction, ELISA permits the highly sensitive and selective quantitative/qualitative analysis of antigens, including proteins, peptides, nucleic acids, hormones, herbicides, and plant secondary metabolites. This specific immune response plays an important role in protecting the body from invaders such as pathogens and toxins. The basic facts about ELISA and its practical use for measuring plant secondary metabolites are described in this review.ĮLISA is based on the concept of antigen–antibody reactions, representing the chemical interaction between antibodies produced by the B cells of leukocytes and antigens. However, ELISA exhibits several advantages over such techniques because of its simplicity, selectivity, and sensitivity. Thus far, various analytical methods have been developed for such purposes, mainly based on high-performance liquid chromatography (HPLC). Meanwhile, simple, selective, and sensitive analytical techniques are also required in pharmacodynamic studies for monitoring effective concentration, side effects, and metabolism, leading to a better quality of life for patients. In addition, Cragg and Newman recently reported that 34% of the currently used drugs originate from natural products. Quality control of these commercial products containing secondary metabolites is crucial as the quality directly affects their potential activity. Because of their diverse functions, there has been a dramatic increase in their demand in pharmaceuticals, cosmetics, and pesticides, as well as in food additives. Plant secondary metabolites are plant-produced organic compounds that play an important role in the defense of plants against herbivores, pests, and pathogens, as well as in their adaptation to the environment, although they are not directly involved in the growth and development of organisms. Currently, ELISA has a higher number of immunoassays compared to RIA. However, radioactive labels have been gradually replaced with enzyme labels because of safety concerns associated with radioactivity since the study by Avrameas in 1969, who coupled antigens or antibodies and enzymes using glutaraldehyde. Since the development of radioimmunoassay (RIA) in 1960, there has been a rapid increase in immunoassay techniques using radioactive labels. This review article describes ELISA and its applications for the detection of plant secondary metabolites. In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin, a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977.
Since the principle of immunoassays is based on specific antigen–antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis.
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