In 1975, Kohler and Milstein reported (1) that immortal cell lines secreting antibody of a single specificity could be produced by the artificial fusion of splenocytes derived from an immune mouse and tumour cells derived from a murine myeloma. They called these cell lines hybridomas and the product from them monoclonal antibodies. The development of monoclonal antibodies opened up huge possibilities in all areas of antibody use because reagents could be created with specificity to a single domain (epitope) on the target

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

substance. Additionally, antibodies could be generated to compounds that had previously been regarded as impossible when using conventional serum production. Monoclonal antibodies should, however, be seen as complementary to those derived from animal serum because each has its place in immunochemistry. The unique specificity, defined affinity, and avidity of the monoclonal antibody are very desirable when looking at cell surface markers or single epitopes on a viral protein. In contrast, the broad specificity of polyclonal antibodies is a characteristic that may be desirable when screening for multiple strains of a virus or in techniques such as immunoaffinity purification.

The techniques for generating hybridoma cell lines have not changed much from the early work of Kohler and Milstein. Original work (2) used mice as donor animals and the mouse myeloma line NS-1 or its derivative NS-O (see Note 1) as the fusion partner. The murine system is probably still the most prevalent today although rats may be used for some antigens. Most murine hybridomas are produced from the physical fusion of spleen and tumour cells using low-speed centrifugation and polyethylene glycol (3). Recombinant hybridoma cells are selectively grown because only they have the necessary characteristics of immortality derived from myeloma cells and a salvage pathway for purine nucleotide (hypoxanthine-guanine phospho ribosyltransferase; HPRT) inherited from the splenocytes (see Note 2). Unfused spleen cells have a limited natural life span in tissue culture, and the myeloma cell line lacks the purine nucleotide salvage pathway that is necessary for survival in the presence of the purine biosynthesis inhibitors (Aminopterin) contained in the selective medium (hypoxanthine aminopterin thymidine; HAT).

Techniques such as electroporation and transfection have successfully been used for hybridoma production, but are much less commonly used than cell fusion assisted by polyethylene glycol.

Hybridomas may be unstable because the techniques used to create them are fairly crude and may lead to a loss of cellular and genetic integrity. Instability is characterized by cell death after a few divisions or change in specificity. It is vitally important to test the hybridomas repeatedly to ensure that loss of specificity or abil ity to secrete has not occurred. The desirable qualities of monoclonal antibodies hinge on the fact that they are the products of a cell line derived from, a single parent clone. To ensure that this is the case it is important to aggressively clone the cells until a cloning efficiency of close to 100%, based on the quality of the antibody is achieved.

The vast majority of hybridomas generated in laboratories are destined to be discarded because they will not have the desired qualities of antibody specificity, growth characteristics, or cloning ability required. In most cases, it is more practical to derive a new cell line rather than try to continue with one that is less than ideal. It is very important to have in mind the qualities of the cell line that are required along with the characteristics of the antibody that are needed before embarking on hybridoma production.

Other species of hybridomas, including human, have been produced but are generally created by the use of viruses conferring cellular immortality. Artificial immunization of the donor is often not practical or ethical and so cell lines are often derived from peripheral lymphocytes obtained from individuals naturally immune to the target substance. Some human monoclonal antibody secreting cell lines have been derived from spontaneously occurring myelomas, but this line of approach frequently is unrewarding as the probability that the antibody will be one of interest is remote.

Nonsecretory myeloma fusion partners with defective purine nucleotide biosynthesis pathways do now exist for a number of species, including humans, and so hybridoma production by cell fusion using polyethylene glycol (PEG) is now a possibility.

A number of non-hybridoma techniques have been developed for in vitro antibody production; a review of recombinant antibody production is covered in Chapter 6.

Hybridoma production can be broken down into four processes, immunization of donor animals, cell fusion, cell selection, and expansion. Each of these stages is important for the quality of the final product. Antigens used to immunize animals must be representative of the target substance (see Note 3) or the likelihood of producing cell lines with the correct specificity is remote. Cell fusions must generate hybridomas but steps must be taken to ensure that neither too few nor too many are generated. Cell lines must be selected using strict criteria to ensure that desirable qualities in both cell growth and stability are present along with the specificity, avidity and affinity required for the final testing format. It is always advisable to screen primary cell lines using the assay format envisaged for the final test (see Note 4). Monoclonal antibodies may perform perfectly well in one assay format but may not for one reason or another convert to another.

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