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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.

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.

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.

Transgenic mice

Transgenic mice can be generated from embryonic stem cells (ESCs). ESCs are cells that correspond to the inner cell mass of the very early vertebrate embryo (blastula stage) that will generate the embryo. These cell can be cultivated in cell culture and transfected with DNA encoding for a gene and regulatory regions, which control the expression of the gene. In some cells the DNA will integrate into the genome, either at random or in a specific region by a process called homologous recombination. The ESCs in which the DNA has integrated can be clonally expanded using antibiotic selection, because the DNA confers resistance against the antibiotic. These cells will then carry a transgene.

When these cells are transplanted into an early embryo at the blastula stage, they will contribute to the embryo and to the germ line. Some of the offspring of these mice, which are a mix of cells with and without the transgene (chimeras), will carry the transgene in all the cells of their body.

Transgenes can be very simple so that for example just every cell in the animals body is expressing a green fluorescent protein. However, nowadays often very complicated expression systems are used, which allows to switch genes on or off in specific cell types of the body, so called conditional transgenes.

One widely used system is the CRE/ loxP system. The CRE recombinase is the switch (on or off) and is itself under the control of a DNA sequence (promotor), which leads to CRE expression only in specific cell types. Additionally, the activity of the CRE can be controlled by the presence or absence of a small molecule (e.g. tamoxifen). This system allows to switch on or off gene expression in specific cells at specific time points of the animals life.