Technique PrinciplesImmunoassays use antibodies (Ab) from natural immune systems to quantify biomolecules. Ab are Y-shaped, blood proteins that are produced by B cells to stop intruding bodies called antigens (Ag). Ag can be viruses, bacteria, or any foreign biomolecule or chemical. When Ag invade a host, they are metby B cells. The B cells recognize a single epitope on the Ag and bind to it. They then produce Ab, often a type of Ab called immunoglobulins (IgG), to immobilize the Ag so phagocytes recognize and consume them. The B cells are thenceforth programmed to make only one type of IgG that is specific to the epitope of the Ag they first encountered. Due to the specificity of the IgG, they are used in most immunoassay techniques. The structure of the IgG is the reason for its variable specificity. It consists of two Fabregions that are antigen-binding sites. These recognize one epitope of one type of Ag. This region is different for every Ag-specific IgG. The Fcregion is the same on all antibodies of a given species. Figure 1There are two methods for producing Ab for immunoassays: polyclonal and monoclonal. Polyclonal Ab are collected from laboratory animals. The procedure requires the animal to be inoculated with Ag. Some time is allowed for incubation before blood is collected. Often a booster inoculation is performed before more blood is collected. The serum is separated from the blood cells by centrifugation and the soluble IgG molecules are purified. Polyclonal Ab vary depending on the epitope their B cells bound to and exhibit limited specificity. Monoclonal Ab are collected in the same way, but, after purification, the Ab are fused with myeloma cells to form a hybridoma line of cells that is self-perpetuating. The hybridoma cells are diluted to a single cell density where one cell is used to grow an entire culture on medium. The Ab produced from this culture have high specificity to a single epitope because they are all clones.