ELISA Detection

2023-12-21 Hits(218)

1.ELISA (enzyme-linked immunosorbent assay)

A quantitative analytical method used to identify the presence and concentration of molecules in biological fluids that shows antigen-antibody reactions by color changes obtained using enzyme-linked couplings and substrates, commonly known as enzyme-linked immunosorbent assays. Molecules in lower concentrations, such as peptides/proteins, vitamins and drugs, hormones, exhibit high levels of specificity to antibodies or antigens developed for them. Because it's almost impossible for antibodies to bind to molecules other than their own antigens. Therefore, when we know that an antigen is specific for a substance, we can identify the type and number of its antibodies, and when we have antibodies, we can find its specific antigen and the number of antigens by this method.


2. The Experimental Principle of ELISA Method

Tubes and microporous plates made of rigid polystyrene, polyvinyl chloride and polypropylene are commonly used as solid phases in ELISA. The microplates used must be able to properly adsorb antigens and antibodies, but not components in other phases.

Enzymes that can be used in ELISA include galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase. Alkaline phosphatase can be stored with its coupled sodium azide at 4°C. Alkaline phosphatase and P-nitrophenyl phosphate are used as substrates and are safe in tablet form, producing yellow in positive reactions. For peroxide couplers, with 5-aminosalicylic acid and n-phenylenediamine as substrates, brown is considered a positive reaction. If galactosidase is used, the sample must be read in the fluorometer. The enzyme-substrate reaction usually completes in 30-60 minutes and can be stopped using sodium hydroxide (NaOH), hydrochloric acid (HCL), or sulfuric acid (H2SO4). Depending on the properties of the conjugate used, the results are read on a spectrophotometer at 400-600nm.


3.Types of ELISA

(1) Direct ELISA: suitable for the determination of the amount of high molecular weight antigen. The surface of the test plate is directly coated with antibodies or antigens. Enzyme-labeled antibodies or antigens make measurements possible. Wash after incubation to remove unbound antigens or antibodies from the medium. The appropriate substrate is then added to the medium to produce the signal through coloration. Measure the amount of antigen or antibody the signal reflects.

(2) Indirect ELISA: The reason why it is called indirect method is that the antigen to be tested is not separated by the primary antibody but by another antibody placed in the culture medium. For example, diseased serum is added to the antigen-coated pore, incubated in a petri dish, and in order to make the antigen-antibody complex visible, it is necessary to add a secondary antibody that recognizes the antibody in the serum and is labeled with an enzyme. The substrate of the enzyme is then added to the medium to produce color and the concentration is measured.

(3) Sandwich ELSIA: The sample is added to the microporous plate coated with antibodies, and then the plate is incubated for a period of time and washed. Unbound antigens are washed away and cannot be removed when specific antigens that bind antibodies are found. After the washing step, antibodies labeled with antigen-specific enzymes are added and incubated. After incubation and washing, if there are antigens in the medium, these cannot be removed because the enzyme-labeled antibodies bind to the antigen. In order to reveal the activity of the enzyme, the enzyme substrate is added to the medium to ensure staining. Coloring indicates a positive result, while not coloring indicates an enzyme deficiency or a negative result, and because the associated protein is sandwiched between two antibody molecules, the method is known as a sandwich sandwich ELISA. The sensitivity of sandwich ELISA is 2-5 times that of other ELISA methods.

(4) Competitive ELISA: The surface of the hole is coated with antigen-specific antibodies or antibody-specific antigens. The sample to be tested and the labeled antigen or antibody are placed into the well at the same time, and the labeled and unlabeled antigen (patient antigen) or antibody molecules compete with each other to bind to the antibody or antigen in the well. After the enzyme substrate is added to the wash hole, the resulting coloring can be quantitatively determined. The concentration of analyte is inversely proportional to the resulting color development intensity. When the amount of antigen or antibody analyzed in the serum is low, high absorbance is obtained, while large quantities produce low absorbance.


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