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Mice are immunised, usually three or four times over the course of 3–4 months, by

the intraperitoneal route using antigen mixed with an appropriate adjuvant (Fig. 7.5).

Test bleeds can be taken to monitor the immune status of the animals. Once the mice

are sufficiently immune they are left for 2–3 months to ‘rest’. This is important as the

cells that will be used for the hybridoma production are memory B cells and require

the rest period to become quiescent.

Mice are sacrificed and the spleens removed; a single spleen will provide sufficient

cells for two or three cell fusions. Three days prior to cell fusion the partner cell line

NS-0 is cultured to provide a log phase culture. If rat hybridomas are to be made then

the fusion partner Y3 or its derivative Y0 can be used. Cell fusions can be carried out

by a number of methods but one of the most commonly used is fusion by centrifuga-

tion in the presence of polyethylene glycol (PEG). Then 26 106 cells of spleen and

fusion partner are mixed together in a centrifuge tube. A quantity of PEG is added to

solubilise the cell membranes and the fusion carried out by gentle centrifugation. The

PEG is removed from the cells by dilution with culture medium and the cells placed

into 96-well tissue culture plates at a cell density of 10 105 per well. From experi-

ence, these cell numbers will produce only a single recombinant cell capable of

growth in each well. Fusion partners are required to have a defective enzyme pathway

to allow selection after cell fusion. NS-0 lacks the enzyme hypoxanthine-guanine

phosphoribosyl transferase (HGPRT) which prevents it from using a nucleoside

salvage pathway when the primary pathway is disabled by the use of the antibiotic

aminopterin. The tissue culture additive HAT which contains hypoxanthine

aminopterin and thymidine is used to select for hybridomas after cell fusion. They

inherit an intact nucleoside salvage pathway from the spleen cell parent which allows

Discard

negative cells

Immunise
mice

Cell fusion with PEG

Test hybridomas

Clone positive cells

Establish cell banks

Harvest

spleen cells

Harvest

myeloma cells

Grow

myeloma cells

Grow cells

for antibody

production

Fig. 7.5Monoclonal antibody production.

275 7.2 Making antibodies