A Simple Rapid Method for Production of Viral Antibody in Mice. ∗

Abstract

A technic for production of antibody in mouse peritoneal fluid was described by Munoz(1) utilizing intraperitoneal inoculations of egg albumin or bovine serum albumin mixed in Freund's adjuvant. Ascites production was induced, however, in only 1/2 the animals tested. Both Herrmann and Kasel and their associates (2,3) used ascites fluid from immunized mice as a source of viral antibodies. Considerable quantities were induced by intraperitoneal injection of Sarcoma 180 cells by the first group and bacterial-adjuvant mixture(4) by the latter. A modification of these methods utilizing Ehrlich tumor cells has proved consistently reliable, required less time or fewer injections and is here described in detail.

Each group of 8 mice, 4 test and 4 control animals from the same litter, was kept together from birth to 21 days of age when the experiment was begun. Undiluted viral infected fluids removed from monkey renal cell cultures maintained on BAF medium(5) with 5% horse serum served as antigens. Each mouse was injected intramuscularly in the hind-quarter with 0.25 ml of viral antigen mixed with a rapid to and fro movement in the same syringe with 0.25 ml of Freund's complete adjuvant (Difco Laboratories). A second dose was given in the same manner one week later. One week after the second inoculation each animal received 0.25 ml of the viral antigen without adjuvant intramuscularly and 0.1 ml was administered slowly by the intravenous route. At the same time each mouse was given intraperitoneally 0.1 ml of undiluted ascitic fluid from mice previously inoculated by the same route with Ehrlich's ascites tumor cells. Approximately 1–5 × 10⁷ cells were injected and 7 days later the ascitic fluid was harvested aseptically with needle and syringe.

Table I summarizes the results with several different viruses. Following immunization it is evident that neutralizing antibodies developed to virtually the same titer in both the serum and ascitic fluid. This has been observed by others for both neutralizing and HI antibodies(2,3,6). It will be recalled that by immunoelectrophoretic technics mouse peritoneal fluid has been found to be similar in composition to mouse serum and that egg albumin antibodies in ascitic fluid have been found in the gamma globulin fraction(7). It should be noted that the pooled ascitic fluids from one group of control animals (Group 6) contained a very low level of neutralizing activity. No explanation for this finding is apparent. The failure to induce formation of adenovirus antibodies probably was due to the low concentration of the viral antigen.