Determination of Antibody Class

Knowledge of antibody class and subclass is a great help in determining the strategy of purification. Unless special immunization and screening procedures have been used, the most frequent antibodies encountered will be IgM and IgG. Hybridomas secreting IgE are rare (Bottcher et al., 1978; Eshhar et al., 1980; Liu et al., 1980), and hybridomas secreting IgA are usually only obtained when the lymphocytes for fusion are from gut-associated lymphoid tissue (Komisar et al., 1982). If the screening procedure uses staphylococcal protein A or streptococcal protein G (see Sections 5.3.5, 8.10.3 and 8.10.4), it is unlikely that classes other than IgG will be detected. IgM antibodies are more unstable, have lower affinity, are more cross-reactive and more prone to nonspecific effects than other classes. They are generally best avoided unless there is no other choice.

There are now numerous commercially available kits for the isotyping of mouse monoclonal antibodies. Many are based on ELISA, either in 96-well trays or dipsticks. In recent years, competition has tended to bring the price of these kits down to a more reasonable level, and it is no longer cost-effective to produce one's own typing sera. However, it may occasionally be useful to perform Ouchterlony analysis, and for this reason the method is outlined in the following section.

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