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Immunization (Generation of antibodies)

An antigen can be almost any molecular substructure (i.e. part of a molecule). Antibodies are proteins, which specifically bind to an antigen and are produced by specialized white blood cells (B-cells) of the immune system upon contact with foreign (i.e. not self) antigens.

This defense mechanism against pathogens can be exploited to produce antibodies against any molecule of interest. Repetitive immunization with an antigen will generate an immune response and lead to the production of many B-cells that produce vast amount of antibodies directed against the antigen. These antibodies can be used in the laboratory to detect specific antigens (e.g. pregnancy test).

The serum (i.e. antiserum) of the blood of the immunized animal contains the antibodies, which can be used for many different techniques. Antisera are often called polyclonal antibodies.

It is also possible to isolate the antibody-producing B-cells from the animals to fuse them with a cancer cell line. The cell hybrids are called are called hybridoma. The technique also allows selection of successfully fused B-cells, which produce the antibody of interest, and indefinite expansion in cell culture. As the hybridoma secrets its antibody into the extracellular environment, the cell culture supernatant contains the antibody of interest. Because a hybridoma secrets one type of antibody originating from one B-cell, these antibody preparations are called monoclonal antibodies.

Similar to antisera, monoclonal antibodies can be used for various techniques.

Fluorescence activated cell sorting – FACS

Is a method to isolate individual cells based on optical features of the cells.

The physical background of each of the steps of the method is rather complicated and won’t be explained here. Briefly, the main steps are hydrodynamic focusing of the cells leading to a stream of fluid in which the cells are aligned one after the other, controlled break up the flow of the fluid into droplets containing the cells, passing the droplets through several laser beams, detecting the emitted signals in real time and sorting the droplets according to this information. The drops are sorted into different collection vessels by applying an electrical field to them, which allows to control the direction of movement of each of the droplets.

Most of the times the cells are stained with antibodies (see IHC/ICC/IF) and then sorted according to the fluorescence of the antibodies. Often antibodies that recognize antigens on the cell’s surface are used, allowing staining and sorting of living cells. Obviously, living cells are much more versatile regarding downstream applications compared to fixed (dead) cells.

Polymerase chain reaction – PCR

PCR allows to amplify specific DNA sequences in an exponential manner and was developed 1983 by Kary Mullis. Because the technique revolutionized molecular biology, he consequentially received the Nobel price 10 years later for this remarkable invention.

The structural building blocks of DNA are the so called nucleotides, which are composed of a purine (Adenine, Guanine) or pyrimidine (Thymidine, Cytosine) base, a sugar and a phosphate group. The nucleotides of each strand are covalently linked by phosphodiester-bonds. DNA is composed of two anti-parallel, complementary strands, which means that the information (i.e. sequence) is present in both strands, because A pairs with T and G with C via hydrogen bonds.

DNA-Polymerases are enzymes, which synthesize the complementary strand in 5’ – 3’ direction (these are the numbers of the C-Atoms of the sugar). They need a single stranded DNA template and a small piece of complementary strand called primer as starting point, which binds with high sequence specificity. Because DNA consists of two strands, each of them can be used as template for DNA replication.

PCR is a simple repetitive sequence of separating the complementary DNA strands by heat (denaturation), letting the primers (one primer for each of the two strands) bind to the target sequence at a lower temperature (annealing) and de novo synthesis of the respective complementary strand starting from the 3’-hydroxylgroup of the respective primer (extension). Because DNA polymerases from organisms that live at ambient temperatures are destroyed at higher temperatures (denaturation step), heat-stable DNA polymerases from thermophilic bacteria (e.g. Thermus aquaticus) are used to avoid having to add new enzyme after each cycle.

These three steps are repeated for 25 to 40 times, which lead to a doubling of the PCR product in each cycle.