1. It is absolutely essential that myeloma cells are resistant to 8-azaguanine and, therefore, are not capable of producing Hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and using hypoxanthine contained in after-fusion selective hybridoma medium. This can be achieved by growing cells in a media containing 8-azaguanine and HT. Resistance to 8-azaguanine results in the elimination of all HGPRT+ revertants, whereas HT preselection leads to optimal survival and growth of primary hybridoma clones in HAT after-fusion medium.

2. The unused splenocytes can be aliquoted, cryopreserved, and, upon careful thawing, utilized for future cell fusions. This could be a very valuable resource to improve the specificity, sensitivity, isotype, and other parameters of the desired antibody, including changing the origin and even species specificity of the fusion partner.

3. In this chapter, we are not attempting to describe typical screening protocols — these differ very significantly between different hybridoma projects, based on the objectives and resources of each research group. The most important general rule here is to build primary and secondary screening protocols exactly in accordance with the intended application of the desired MAb and, if possible, perform screening procedures using experimental techniques closely linked to the intended application. In other words, if you are planning to develop a MAb for immunohistochemistry staining, it would be not a good idea to use ELISA for the selection of hybridoma clones. Although tempting for experimentators who are new to the hybridoma field, these seemingly straightforward and inexpensive strategies rarely work well in the long run, resulting in selecting numerous clones with unfavorable parameters in the desired application.

4. The objective of recloning is not only to ensure the clonality of resulting subclones and ensuing hybridoma mass population used to produce and manufacture large amounts of the desired MAb, but also to select for the most stable, quickly growing, and highly producing MAb subclones. This approach ultimately results in generating durable and reliable hybridoma cell banks that can be used for large-scale production on highly active MAb immunoglobulins.

5. As discussed in Subheadings 1. and 4., generating MAbs in tissue culture is a viable and increasingly popular alternative to growing ascites in BALB/c and nude mice. The former approach does not require animal use and allows the full realization of the potential of MAbs by avoiding nonspecific antibody contaminants.

6. Similarly to the immunization and screening protocols, every research group uses its own approach and methodology for purifying MAbs. Therefore, in this paper we are not attempting to present a detailed purification protocol that would be acceptable for all experimental models and objectives.

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