Mice should be obtained from a reputable source and should be maintained under standard conditions as required by local animal welfare legislation. Female mice can be housed together and it is usual to indelibly mark individuals by tattoo, ear punch or electronic chip so that each animal and its immune response can be monitored.

1. Prepare antigens at approx 1 mg/mL in physiological saline.

2. Inject the mice intraperitoneally with 0.05 mL of antigen mixed with an appropriate adjuvant on d 0, 14, 28, and 44. Take a test bleed on d 51 and assess the serum for the presence of circulating antibody to the antigen.

3. If the immune response is good (see Note 8) rest the animals for a minimum of 8-10 wk before cell fusion work. If the immune response is poor give another immunization on d 61 and assess the circulating antibody level on d 68.

4. Prior to carrying out a fusion, give the mouse with the highest antibody titer a booster dose of 0.05 mL of antigen without adjuvant intraperitoneally. Three days later, kill the mouse by cervical dislocation and remove spleen aseptically.

5. If all mice have responded well to the immunizations give all of them a booster dose, harvest the spleens and cryogenically store the splenocytes for use at a later date (see Note 9). Store the spleen cells cryogenically as described in Subheading 3.6.

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