Initiation and maintenance of B-hybridoma cell lines

2.7 Initiation and maintenance of B-hybridoma cell lines

2.7.1 Original method for the propagation of hybridomas

An outline of this procedure is included for historical reasons; however, it is no longer allowed in the UK owing to changes in the animal welfare regulations.

Freshly isolated hybrid cell cultures often grow slowly and are less tolerant of low cell densities than their plasmacytoma parent. The volume of the cell culture must be expanded slowly, at a rate that can only be determined empirically because hybrid lines show different growth rates. Colonies or cloning wells should be transferred to a maximum of 0.2–0.5 ml of medium (again with a feeder layer if necessary) and diluted with an equal volume of fresh medium as the pH indicator dye just begins to turn an orange-yellow. Hybridoma lines grown up to stationary phase in static flasks or spinner culture vessels can produce up to 1 µg/ml of antibody protein. Although the antibody is pure, the spent medium contains many other serum proteins. Large amounts of hybridoma-derived antibody may be prepared by injecting these tumorigenic lines into histo- compatible (or immunoincompetent) mice.

2.7.2 Alternative method for the propagation of hybridomas

Selected hybridomas in HT medium may be expanded from a 96-well microtitre plate to a 24-well plate and finally a 75-cm 2 flask. Hybridomas may also be propagated in tissue culture using hollow-fibre technology, e.g. MAbMAX, Bio Whittaker; Harvest Mouse, Serotec; Technomouse, Northumbria Biologicals. This approach avoids the necessity of ascites production.

2.7INITIATION AND MAINTENANCE OF B-HYBRIDOMA CELL LINES

2.7.3 Production of monoclonal antibodies from a hybridoma line within in vitro culture

MATERIALS Mice (histocompatible with hybridoma or nude, athymic) Hybridoma line from in vitro culture Pristane (2,6,10,14-tetramethylpentadecane)

METHOD

1 Inject 0.5 ml Pristane into the peritoneal cavity of each mouse.

2 After 7 days, inject 10 7 hybridoma cells i.p. into each mouse.

Most hybridoma lines will produce solid tumours or ascites within 2–3 weeks.

3 Use a syringe and 19-gauge needle to drain off the ascitic fluid. Clarify the ascitic fluid by centrifugation (500 g for 15 min at 4°C).

4 If desired, screen the ascitic fluid from individual mice by electrophoresis and store those samples showing a prominent peak of paraprotein in the g-globulin region (see Section 3.7).

5 Repeat steps 3 and 4 for the lifetime of the mouse. TECHNICAL NOTES

• Ascitic fluid often contains up to 1 mg/ml of specific antibody protein. There are, of course, other proteins, including immunoglobulins of unknown specificity. • The serum of these tumour-bearing mice also contains large quantities of hybridoma-derived antibody. • It is inadvisable to maintain a hybridoma by serial passage in mice because of the risk of accu- mulating non-secreting cells. Instead, inject large batches of mice with recently cloned cells from in vitro culture.

• Immunoincompetent nude or severe combined immunodeficient (SCID) mice will also be required if the hybrid line was derived from a mouse–rat fusion. Some investigators prefer to immunize rats, rather than mice, for fusion, as it is possible to obtain significant volumes of anti- sera in test bleeds prior to fusion.

• Passage through an animal can be helpful for a valuable monoclonal, to remove mycoplasma infection.