Indirect ELISA is an extremely sensitive and flexible procedure. After washing to remove any unbound antibodies, a chromogen is added.The presence of the enzyme converts the substrate into a colored end product. If the antigen is present, then the antibody will bind. An antibody that is specific for a particular antigen, and is conjugated to an enzyme, is added to each well. In direct ELISA, antigens are immobilized in the well of a microtiter plate. There are several types of ELISA, based on differences in the format of detection and general workflow. Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer. ELISAs that utilize a fluorogen are termed fluorescent enzyme immunoassays (FEIAs). In some ELISAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase, for which appropriate substrates are readily available. In ELISAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. Enzyme-substrate reaction allows the antigen to be visualized or quantified. There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. ELISA differs from western blot in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane. In 1971, Peter Perlman and Eva Engvall developed an Enzyme-linked immunosorbent assay (ELISA or EIA). The higher the sample antigen concentration, the more antigen-antibody complexes are formed, leaving fewer free antibodies to bind to the competing antigen from the plate, therefore generating weaker signals. In competitive ELISA, the antigen and the primary antibody are pre-incubated and added to the plate that is coated with the same antigen. Sandwich ELISA involves two antibodies, one coated on the plate to capture the sample antigen, while the other enzyme-conjugated antibody binds to the specific antigen-epitope forming the sandwich. While for indirect ELISA, the attached antigen captures the sample primary antibody, to which the conjugated secondary antibody binds. The detection antibodies that bind to the antigen or target molecule are conjugated with specific enzymes which, upon substrate addition, produce measurable signals.ĭepending on the capture and detection methods, there are four major types of ELISA.ĭirect ELISA uses an enzyme-conjugated primary antibody for detecting the sample antigen coated on the plate. Enzyme-linked immunosorbent assay or ELISA is a labeled immunoassay with wide applications in detecting and quantifying analytes like antibodies, peptides, glycoproteins, hormones, and viruses.
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