I. Enzyme-Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) is a quantitative analytical method that utilizes color changes generated by enzyme-conjugated substrates to detect antigen-antibody reactions, commonly employed to identify the presence and concentration of molecules in biological fluids. This technique demonstrates high specificity for low-concentration molecules such as peptides/proteins, vitamins, drugs, and hormones through their developed antibodies or antigens. Since antibodies rarely bind to molecules other than their specific antigens, ELISA enables two key applications: when the antigen's specificity to a particular substance is known, we can determine the type and quantity of its corresponding antibodies; conversely, when antibodies are available, this method allows identification of their specific antigens and quantification of antigen levels.

II. How Does the ELISA Detection Method Work?

ELISA typically utilizes rigid polystyrene, polyvinyl chloride, or polypropylene tubes and microplates as the solid phase. These microplates must effectively adsorb antigens and antibodies while preventing adsorption of components from other phases.

Enzymes suitable for ELISA include galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase. Alkaline phosphatase can be stored at 4°C together with its coupled sodium azide. Alkaline phosphatase and p-nitrophenyl phosphate serve as substrates, provided in safe tablet form, producing a yellow color in positive reactions. For peroxidase conjugates, 5-aminosalicylic acid and o-phenylenediamine are used as substrates, with a brown color indicating a positive reaction. If galactosidase is employed, the samples must be read using a fluorometer. The enzyme-substrate reaction is typically completed within 30-60 minutes and can be stopped using sodium hydroxide (NaOH), hydrochloric acid (HCl), or sulfuric acid (H₂SO₄). Depending on the characteristics of the conjugate used, the results are read on a spectrophotometer at wavelengths between 400-600 nm.

III. Types of ELISA Assays

(1) Direct ELISA:This method is suitable for measuring the quantity of high-molecular-weight antigens. Antibodies or antigens are directly coated onto the surface of the assay plate. Enzyme-labeled antibodies or antigens enable measurement. After incubation, washing is performed to remove unbound antigens or antibodies from the medium. An appropriate substrate is then added to the medium, generating a signal through color development. The measured signal reflects the amount of antigen or antibody present.

(2) Indirect ELISA:This method is called "indirect" because the antigen being measured is not captured by the primary antibody but rather by another antibody introduced into the medium. For example, diseased serum is added to antigen-coated wells, and the plate is incubated. To visualize the antigen-antibody complex, a secondary antibody—which recognizes the target antibody in the serum and is enzyme-labeled—is added. The enzyme substrate is then introduced into the medium to produce color, and the concentration is determined.

(3) Sandwich ELISA:The sample is added to an antibody-coated microplate, which is then incubated and washed. Unbound antigens are removed, while any antigens specifically bound to the antibodies remain. After washing, an enzyme-labeled antibody specific to the antigen is added, followed by another incubation. If antigens are present in the medium, they cannot be washed away because they are bound by the enzyme-labeled antibody. To detect enzyme activity, a substrate is added to the medium, resulting in color development. A color change indicates a positive result, while no color change suggests the absence of the enzyme (negative result). Since the target protein is "sandwiched" between two antibody molecules, this method is called sandwich ELISA. Its sensitivity is 2–5 times higher than other ELISA methods.

(4) Competitive ELISA:The wells are coated with either antigen-specific antibodies or antibody-specific antigens. The test sample and a labeled antigen or antibody are simultaneously added to the wells, where the labeled and unlabeled antigens (patient antigens) or antibodies compete for binding to the immobilized antibodies or antigens. After washing, an enzyme substrate is added, and the resulting color intensity is measured to quantify concentration. The analyte concentration is inversely proportional to the color intensity: low antigen/antibody levels yield high absorbance, while high concentrations produce low absorbance.

IV. Advantages and Disadvantages of Different Types of Enzyme-Linked Immunosorbent Assay (ELISA)

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