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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 secretes 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.

Immunohistochemistry

Immunohistochemistry is a method to visualize antigens in tissue section by using 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 of an animal, e.g. a rabbit or a goat, 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 of the blood of the immunized animal contains the antibodies. This antiserum can be used to probe tissue sections to test whether the antigen is present in the tissue section. To visualize the antibodies and consequently also the location of the antigen in the section, the antibody can be labeled in various ways. Most common are fluorophores or enzymes as labels. Usually a so-called secondary antibody is labeled, which detects the antibodies of the antiserum from the immunized animal (the primary antibodies). This results in a sandwich composed of the tissue with the antigen that is bound by the primary antibody, which is in turn bound by the secondary antibody that carries the label for detection. Fluorescent labels can be detected using fluorescence microscopes and enzymes as label can be detected by their enzymatic activity, which is a color reaction.

Cell culture

Cell culture is a method to culture cell lines or primary cells in vitro. Cells in cell culture are accessible and easy to manipulate making experimental setups convenient.

The basic principles of cell culture were already established beginning of the 20th century, when developmental biologists started to culture organs and embryo explants in salt solutions supplemented with body fluids such as amniotic fluid, allantois extract or serum. In the 50ties of the previous century the widespread virology research spurred the development of robust cell culture protocols for mammalian cell lines, which were used to produce viral particles for research and vaccine development.

Cells freshly isolated from the body are called primary cells. The cells that can be isolated most easily from mammals are cells of the peripheral blood. The isolation of cells from tumors and subsequent long term culture or infection of primary cells with tumorigenic viruses lead to the generation of many cell lines, which are virtually immortal and can be cultured much easier than primary cells.

Establishing cell culture conditions was and is still trail and error by just testing out many different ingredients and conditions, and is usually very complex. For a long time the biological reasons for many ingredients that support the growth of a particular cell type were unclear or as for serum the active ingredients were just unknown. However, since the 1950ies we learned a lot about a cells biology and understand much better what a cell need to be happy. Yet, out of the approximate 200 different cell types of the human body only some can be cultured with ease. To reduce experimental variability, there are a lot of efforts to get rid of variable and undefined ingredients such as serum and instead to move towards chemically defined medias in which the concentration of all components, ranging from salts, organic chemicals, amino acids, peptides and proteins, are exactly known

But if you are a scientist nowadays working with mammalian cells, cell culture is in fact a routine job. Basically all media, supplements and dishes can be purchased commercially. For all cell lines and many primary cells robust standard culturing protocols are available.

Enzyme linked immune sorbent assay – ELISA

ELISA is a highly sensitive method to detect antigens in samples by antibodies.

Antibodies that are specifically directed against the antigen of interest can be generated by immunization. To detect the antigen of interest, the sample needs to be attached to a plastic surface (usually a 96-well plate). ELISA plates have an special surface to which antigens (proteins) stick. After the sample is bound to the plate the remaining sticky surface of the plate is blocked by using unrelated proteins like serum albumin. The primary antibody can be used to probe the sample to test whether the antigen is present. To detect the primary antibody usually secondary antibodies are used, which specifically detects the primary antibody. This results in a sandwich composed of the antigen sticking to the plate, the primary antibody bound to the antigen and the secondary antibody bound to the primary antibody. The secondary antibody carries a label, which is an enzyme. The secondary antibody can be visualized by the enzymatic activity, which results in a color reaction.

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.