Immunization volumes should not exceed 0.2 ml when administered subcutaneously or 0.5 ml when administered by intraperitoneal injection. In vitro immunization procedures have been reported to be beneficial when antigen is in limited supply and previous attempts at in vivo immunization have failed. Other advantages of in vitro immunization are the requirement for fewer animals and the immunization period is reduced to 4-5 days 4,5,6 . Freund's incomplete adjuvant has been demonstrated to be as good or better than pristane as a priming agent 1,2,3 . It is recommended that hybridomas be tested for the presence of adventitious viral and mycoplasma agents prior to use in animals.
Viral contamination is common in murine leukemia and transplantable tumor specimens.
Inoculation may result in infection of the animal and spread of disease through the vivarium. Also, lymphocytic choriomeningitis virus has been isolated from tumor cell lines which is capable of producing illness in humans 7 . The second tap should be performed after the mouse has been euthanized. However, in more aggressive cell lines which cause significant morbidity it may be necessary to limit taps to less than this and vice versa for cell lines that are more slowly progressive and cause minimal morbidity, more than two taps may be appropriate. Ascites pressure should be relieved when visible abdominal distention becomes evident, and prior to the development of marked abdominal distention with associated clinical signs of pain or distress.