The substrate for HRP is hydrogen peroxide and results in a blue color change. However, the most commonly used horseradish peroxidase (HRP) and alkaline phosphatase (ALP). There are many substrates available for use in ELISA detection. ĭetection is carried out by the addition of a substrate that can generate a color. ![]() However, this method is qualitative and can determine only the presence or absence of an analyte and not its concentration. This technique can generate a color signal visible by naked-eye, with blue color for positive results and red color for negative results. The latest development, in 2012, was an ultrasensitive enzyme-based ELISA that manipulates nanoparticles as chromogenic reporters. However, some of these techniques do not rely on using enzyme-linked substrates but non-enzymatic reporters that utilize the principle of ELISA. Further advancement in the ELISA technique leads to the development of fluorogenic, quantitative PCR, and electrochemiluminescent reporters to generate signals. The first ELISA methodology involved chromogenic reporter molecules and substrates in generating observable color change that monitors the presence of antigen. Since then, the ELISA method has been used in many different applications and became a routine laboratory research and diagnostic method worldwide. Within the same year, scientists were able to quantify human chorionic gonadotropin in urine by using horseradish peroxidase. The new method was first employed by determining the levels of IgG in rabbit serum. This was done by conjugating tagged antigen and antibody with enzymes rather than radioactive iodine 125. The ELISA was developed by the modification of the radioimmunoassay (RIA). Two different research teams invented the direct ELISA simultaneously by scientists Eva Engvall and Peter Perlman and by Van Weemen and Schuurs. ![]() This article will discuss the basic principles, procedures, and clinical significance of the ELISA. ELISA testing is used to diagnose HIV infection, pregnancy tests, and blood typing, among others. ![]() This interaction is utilized in ELISA testing and allows for identifying specific protein antibodies and antigens, with only small amounts of a test sample. An antigen is a protein that can come from some foreign source and, when bound to an antibody, induces a cascade of events through the body’s immune system. This protein type has specific regions that bind to antigens. An antibody is a type of protein produced by an individual’s immune system. The detection of these products is accomplished by complexing antibodies and antigens to produce a measurable result. This immunological test is very sensitive and is used to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. Enzyme-linked immunosorbent assay (ELISA) is a labeled immunoassay that is considered the gold standard of immunoassays.
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