Hybridoma technology

  • Immunization ( eg mouse)
  • Cultivation of myeloma cells
  • Recovery of B cells ( spleen)
  • Cell fusion
  • Selection of hybridoma cells ( HAT selection )
  • Screening for competent, antibody -secreting cells ( ELISA)

Immunization

The first step in the production of hybridoma cells represents the immunization of donor organisms to gain from them suitable B- cells. For this purpose, usually specially bred mice inbred lines ( BALB / c- line ), since these are hardly susceptible to disease. Furthermore, human, rabbit, rat and goat systems are available. The immunization of the donor organism usually repeated low doses of antigen ( vaccine ) should be administered. Due to the natural immune response B- cells are propagated is formed which secrete antibodies against said antigen. The exact sequence of immunization depends on the present and the organism to be administered antigen and is often only determined empirically. A guideline for the immunization of mice, for example, the administration of 100 micrograms antigen and 300 ul PBS buffer. To increase the chances of success, three or more animals are also used in the rule. Four days after the last immunization, the animal is taken as accumulate in this organ particularly large number of B- cells, the spleen. The extraction of this just ultimately takes place by a density gradient centrifugation. At the same time in myeloma cell cultures are grown that have lost their cell-specific functions (eg, the production of antibodies ). To be particularly suitable cancer cells of the plasma cells have been found, since apoptotic signals are set entirely suspended. They provide after cell fusion that is only for the unlimited proliferation of the hybridoma cells. It has also proved to be advantageous to use myeloma and B - cells from the same organism as a result, the stability of the hybridoma cells is increased.

Cell fusion

Essentially, two methods have been established for cell fusion: (1) the action of chemical agents ( polyethylene glycol, PEG) or (2 ) the treatment means of electric power ( electric fusion). In the first method B cells and myeloma cells are combined in the fusion solution and centrifuged. Because the water present is substantially bound by polyethylene glycol, the cell membranes are brought into close contact with each other, whereby a spontaneous fusion of the cell membranes is achieved. Since PEG exerts a toxic effect on cells that are concentration and exposure time of the PEG solution for the success of the merger of crucial importance. In the electro-fusion the cell membrane is by means of electrical pulses locally " melted " (see, electroporation) to form a fusion of the cell membranes is achieved. In order to support the merger, PEG is added in small amounts in most cases here. After the merger arise among other cells that have two or more nuclei ( heterokaryon ). To become an intact hybridoma these nuclei must merge spontaneously. Because chromosomes are frequently rejected in this process, only a very small part of the hybridoma cells remains stable.

Selection of hybridoma cells

After cell fusion, five cell types are present in the solution:

  • Hybridoma
  • Unfused spleen cells
  • Unfused myeloma cells
  • Fused spleen cells
  • Fused myeloma cells

Order it now to select hybridomas that produce actually (monoclonal ) antibodies, one uses a selective medium in which hybridoma cells are only able to survive. A suitable medium is the so-called HAT- medium containing the chemical substances include hypoxanthine, aminopterin, and thymidine. Hypoxanthine is a precursor of molecules is essential ( purines ), which are required for the construction of the deoxyribonucleic acid ( DNA). However, its conversion to the enzyme HGPRT (hypoxanthine - guanine phosphoribosyl transferase ) is necessary. Since the merger, a myeloma cell line is used, which is specifically lacking this enzyme, or in which it is present in an inactive form, individual myeloma cells in this medium thus not viable. Spleen cells, however, possess HGPRT are for themselves but not viable and die in the culture medium rapidly. Only hybridoma cells can be cultured as they possess the immortality of the myeloma cells and the spleen cells of the HGPRT gene. Aminopterin only serves to block other purine synthesis pathways. Characterized also because the essential de novo thymine can not be made ​​, it must be added to the medium ( see also pyrimidine de novo synthesis). Among the thus selected hybridoma clones but those still need to be isolated which produce the desired antibodies ( screening). This is preferably done by an ELISA ( enzyme-linked immunosorbant assay ) test. The antibodies are incubated doing with that antigen against which this should actually be addressed. A match is thereby detected by an enzymatic color reaction. Are obtained after HAT selection, for example, tens of thousands of clones, it is not uncommon among these only a few hundred cells produce the desired antibodies. Often only a few dozen of them remain stable hybridoma After further cultivation. A part of the positive clones is stored for later use in liquid nitrogen, while the remaining cells are further cultured. These hybridoma cultures achieve yields of up to 1 mg mAb / ml.

in vivo House mouse Phosphate buffered saline Incubation
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