Hybrid clones will appear within 2–3 wk after fusion (seeChapter 46 ). It is of utmost importance that a newly established hybridoma is cloned thoroughly to ensure that the cells growing in the tissue culture are of monoclonal origin and not a mixture of two or more hybridomas. A mixture of cells may result in a gradual decline of specific antibody production because of overgrowth of contaminating hybridomas, until finally the hybridoma of interest is lost. Culture supernatants from the hybridomas should be tested for specific antibody production by appropriate methods. Hybridomas are cloned by limiting dilution using thymocytes as feeder cells as soon as the cell density in the well reaches one-half to two-third confluence.
