Hybridoma production

Wojchowski, D.M., and Sytkowski, A.J. (1986) Hybridoma production by simplified avidin-mediated electrofusion./. Immunol. Meth. 90, 173-177. 

A number of protocols will be described here that have been used successfully with both rat (Y3 and IR983F) and mouse (SP2/0) myelomas to generate MAbs to cellular proteins, recombinant proteins, or peptides based on cDNA sequences. Successful hybridoma production relies on the ability to ... 

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. 

Media basal RPMI 1640 medium requires supplementation with sodium pyruvate, L-glutamine, and penicillin/streptomycin before use. The supplements are supplied as concentrates of 50X or 100X, and the appropriate amounts should be added. Standard tissue culture media for hybridoma production contain 5%, 10%, or 15% fetal bovine serum (FBS see Note 6). Sufficient quantities should be prepared in advance and a sterility check should be performed on them prior to use. 

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. 

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. 

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... 

Micklem, L. R., McCann, M. C., and James, K. (1978) The use of rat mixed thymocyte-conditioned medium for hybridoma production, cloning and revival. J. Immunol. Methods 104, 81-86. 

Kohler G (1981), The technique of hybridoma production, In Immunological Methods, vol. II, Academic Press, pp. 285-308. 

Hybridoma production is the initial step in the production of monoclonal antibodies which is now a major bioengineering industry. There are many books devoted to this topic (e.g. Campbell, 1984, in this series). 

B lymphocytes will be eliminated during continuous culture because these cells have a short life span in culture. Commercially available myeloma cells for hybridoma production have mutations in one of the enzymes of the salvage pathway of purine nucleotide biosynthesis. Hybridoma cells are cultured in medium that forces the cells to utilize the salvage pathway for nucleotide synthesis. The mutated myeloma cells or hybridization products of two myeloma cells will die in this selection medium since they are incapable of nucleotide synthesis under these propagation conditions. However, myeloma cells that have fused to the B lymphocytes derived from the spleen of the immunized animal will have an intact salvage pathway and will survive in the selection medium. Thus, only the B lymphocytes-myeloma hybridomas will survive prolonged culture in the selection medium. 

Successful hybridoma production relies on the ability to (1) generate specific Rcells, (2) fuse them with a myeloma cell line, (3) identify the antibodies that are sought in culture supernatants, and (4) isolate and clone the... 

Persistence is an important attribute of the hybridoma producer fusions can fail for many reasons, and it is essendal not to give up because of early failures. The methods described in this chapter include techniques for (1) preparadon of andgen, (2) immunization, (3) hybridoma production, (4) assaying the MAb-producing hybridomas, and (5) isolation and purification of MAbs. 

A sensitive screening method is a sine qua non for successful hybridoma production and should be developed before starting the experiment. The first screening should take place 5 days after fusion, followed by other inspections every other day. EIA, particularly with POase are used for the screening of antibodies but it should... 

C Milstein, AC Cuello. Hybrid hybridoma production of bi-specific monoclonal antibodies. Immunol Today 5 299, 1984. 

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