In Vitro Immunization

This technique is performed in tissue culture and works well when only small quantities of antigen (1-2 ^g) are available. It also allows the production of antibodies to substances that are toxic in whole animals. There is no immunological processing of antigens so only soluble, simple antigens such as peptides can be used for this approach. A source of interleukins-4 and -5 is required for the method to work and the easiest way of obtaining them is from thy-mocyte-conditioned medium.

3.6.1. Thymocyte-Conditioned Medium

1. Kill two 6-wk-old Balb-c mice and remove their thymus glands asep-tically.

2. Homogenize the tissue to produce single cells and resuspend in 10 mL of RPMI 1640 medium containing 15% fetal bovine serum.

3. Incubate the medium at 37°C/ 5% CO2 for 24 h (see Note 6) and then harvest the supernatant by centrifugation (700g). Store the conditioned medium at -20°C until required.

3.6.2. Immunization

1. Kill a non-immunized balb-c mouse and remove its spleen asepti-cally.

2. Homogenize the spleen to produce single cells, and then resuspend them in 10 mL of the thymocyte-conditioned medium.

3. Add 1-2 ^g of antigen to the cell suspensions and incubate at 37°C/ 5% CO2 for 72 h (see Note 7).

4. Harvest the spleen cell by centrifugation (500g) and use immediately for a cell fusion.

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