Hybridomas are cell lines historically used in the production of monoclonal antibodies. Mammals such as mice are immunized with an antigen of interest, and their antibody-producing B lymphocytes are harvested. The lymphocytes are fused with immortalized myeloma cells to form hybridomas, which are essentially immortal antibody-producing cell lines that can be cultured in vitro. Each hybridoma cell line is derived from a single lymphocyte, so the resulting population of cells is clonal and produces molecularly identical immunoglobulins – monoclonal antibodies.
In 1984, César Milstein and Georges J. F. Köhler won the Nobel Prize in Medicine and Physiology for their 1975 invention of monoclonal antibodies via the hybridoma technology (the Prize was shared with Niels K. Jerne for his theories concerning the development and regulation of the immune system).
Horseradish peroxidase (HRP) is a 44 kDa enzyme found in the roots of the horseradish plant, Armoracia rusticana. Chemically, it is a metalloenzyme because it contains iron (in the form of heme) and an oxidoreductase because it catalyzes a redox reaction (specifically the reduction of hydrogen peroxide and the oxidation of the enzyme’s substrate). HRP works on a number of substrates, including the chromogenic substrates 3,3′-diaminobenzidine (DAB) and 3,3′,5,5′-tetramethylbenzidine (TMB), oxidizing them from colorless (or only slightly colored) forms into intensely colored forms.
In immunohistochemical applications, HRP can be conjugated to an antibody, and the conjugate’s reaction with a chromogenic substrate can be used to detect the presence of the antigen visually under optical microscopy. In other contexts, the substrate may instead be chemiluminescent, and its oxidation gives off a specific wavelength of light that can be sensitively measured. For instance, HRP oxidizes the compound luminol and gives off 428 nm blue light. These chemiluminescent methods can be used detect very small amounts of DNA in a Southern blot, or of RNA in a northern blot.