Horseradish Peroxidase (HRP) is a glycoprotein with a molecular weight of 44000 Da. It’s constituted by a colorless zymoprotein bound with a dark brown ferriporphyrin. The HRP used in enzyme immunoassay contains various isozymes with the so-called “C” isozyme as the main component. Other isozymes have relatively low activity.
HRP shows high specificity for hydrogen acceptor. Besides H202, it only acts on the peroxide of small-molecule alcohols and carbamide. The solid carbamide reagent is more convenient than H202.
Many compounds can act as the hydrogen donor of HRP. The common hydrogen donor substrates used in ELISA are O-Phenylenediamine (OPD), tetramethyl benzidine (TMB), and ABTS. Despite the advantage of high sensitivity and colorimetric convenience, OPD has poor stability after prepared into application solution and is prone to cause denaturation. Instead, TMB is free from these defects. After the colorless TMB reacts with the enzyme, it will appear blue in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color turns yellow. The color changes of the whole process are very obvious to the human eye. And the optical density (OD) can be measured spectrophotometrically. Therefore, TMB is the most common used substrate in ELISA. ABTS, though not as sensitive as OPD and TMB, has relatively low blank value.
HRP is the most widely applied labeling enzyme in immunoassay such as ELISA, WB and IHC. On one hand, HRP is easy to collect and relatively cheap. On the other hand, HRP is stable, and resistant to heat and organic solvent. Moreover, its activity reduces little after it conjugates with antigen or antibody.
The sodium periodate treatment is the common method for HRP to label monoclonal and polyclonal antibody. The mechanism is as follows. The glycosyl of HRP is oxidized by sodium periodate into aldehyde group which then binds with the amino group of the antibody IgG, forming Schiff reagent. To prevent the oxidized aldehyde group of HRP from conjugating with the amino group of its own, dinitrofluorobenzene (DNFB) is used to block the amino group before sodium periodate is added. After the oxidation reaction, sodium borohydride is used to stablize the Schiff reagent.
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