The very first Nobel Prize in Physiology or Medicine was awarded in 1901 to Emil von Behring for his work on serum therapy and the discovery of diphtheria antitoxin in 1890 (The Nobel Prize in Physiology or Medicine 1901). By injecting tiny doses of weakened bacteria into animals, Emil von Behring and collaborators discovered molecules in the blood that could neutralize the bacterial toxins. When transferring the blood sera to infected animals the symptoms disappeared and the animals were cured. The neutralizing molecules were logically called antitoxins, however, today these neutralizing molecules are known as antibodies.
Antibodies are the body’s natural defense against pathogens and unknown substances that enters the body. The body has a natural pre-immune repertoire of antibodies. An enormous number of antibody variants can be created by an ingenious rearrangement of gene segments. When a foreign substance enters the body, the antibody catalogue is scanned by a special class of white blood cells, called B lymphocytes, to locate the best binding antibody. This antibody can then be produced by the B lymphocytes. When an immune response is elicited, a number of B lymphocytes are activated that will produce antibodies to different parts of the foreign substance, the antigen. The specific area where an antibody binds the antigen is called an epitope, and the generated pool of antibody variants may bind to different epitopes with varying strength. The strength by which the antibody and antigen bind to each other is called affinity.
Due to antibodies’ ability to bind a vast number of different molecules, their naturally good affinity, and their ability to recruit various cells and molecules to destroy pathogens, they are a natural choice for biotechnological and therapeutic applications. The first methods to produce antibodies for applications used animals which were exposed to the antigen in order to produce antibody containing serum. The antibodies produced with this method are called polyclonal as they come from several clones of B-cells and thus bind several epitopes with different affinity. It was not until 1975 that a method for producing antibodies from a single clone was developed through the work of Georges J.F. Köhler and César Milstein (The Immune System: In Defence of our Lives). Fusing one antibody producing B-cell with a cancer cell, resulted in an immortal hybrid cell, a hybridoma, with the ability to multiply and produce the specific monoclonal antibody indefinitely. Since monoclonal antibodies typically target one epitope, with one affinity, and can be produced in unlimited quantities they are ideal for standardized procedures that are essential for therapeutic and diagnostic applications. Polyclonal antibodies on the other hand have a different set of strengths; since polyclonal antibodies target several epitopes they are less sensitive to changes in the antigen, may produce a higher signal, and are useful when the antigen is unknown. Polyclonal antibodies are extensively used for research purposes. In general, both monoclonal and polyclonal antibodies may be successfully used in both therapeutic and biotechnological applications, although they may be more commonly used in one area or the other.
Antibodies are used in a variety of research applications and techniques, such as Western Blot, Enzyme-Linked Immunosorbent Assay, Immunohistochemistry, Immunocytochemistry, Immunoprecipitation, and more. The different conditions in each application and the complexity of biological samples ask for great functionality from the antibodies.
The following pages describe some of the most common techniques.
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