In dogs experimentally infected with Dirofilaria immitis, antibodies were not detected by complement fixation (CF) before the 3rd month, whereas with indirect hemagglutination (IHA) antibodies were detected as early as 2 weeks after inoculation. The overall pattern of a progressive rise and fall in antibody level was basically the same with the CF and the IHA tests and after the 9th month antibodies could not be detected with either test or with any of a number of different antigens. IHA with an acid-soluble protein fraction was the most sensitive test system used. Although this antigen was also the most specific, it reacted in low titers with sera from rabbits infected with Ascaris lumbricoides or Toxocara canis and from humans infected with Schistosoma japonicum or T. canis. The absence of microfilaremia in many persons with filariasis has stimulated investigation of immunological tests which, hopefully, would provide evidence of the infection (Kagan, 1963). Except in a few instances, the antigens used in the above tests were prepared from animal filariae and therefore the tests relied on group specificity (Kagan, 1963). Studies by Pacheco and Danaraj (1963) indicated that the specificity of some of these tests is not restricted to filariae. Moreover, there is evidence that the antibodies demonstrated by these tests are not present throughout the course of a filarial infection (Minning and McFadzean, 1956a). Studies on human filariasis by Lloyd and Chandra (1933) and by Schofield (1957) supported this view and the authors postulated that a relationship exists between detectable antibody and active disease, but not between antibody and microfilaremia. Because of the limited knowledge of the natural patterns of antibody production in the filariases, attempts were made in the present study to: (1) determine antibody levels with several antigens and two tests during the Received for publication 12 April 1965. t Present address: Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, Maryland 20014. * A portion from a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This investigation was conducted under the sponsorship of the Commission on Parasitic Diseases, Armed Forces Epidemiological Board, and supported in part by the Office of the Surgeon General, Department of the Army, and by grants 2E-2 and E1159 from the NIH, U. S. Public Health Service. course of experimental infections of natural h sts of D. immitis, (2) determine whether more than one antibody could be detected by the various antigens used, and (3) evaluate, on a limited scale, the specificity of the test systems used. MATERIALS AND METHODS Subcutaneous inoculations of 200, 70, 20, and 15 infective third-stage larvae of D. immitis were made into four laboratory-whelped pups, respectively. The dogs, 10 to 12 weeks of age when inoculated, were bled from the radial vein before infection, every week thereafter until antibody titers were below detectable limits, and once a month after that. Preparation of worm extracts Adult male and female D. immitis were collected alive from dogs killed at the Jefferson Parish (Louisiana) Rabies Control Center. The worms were separated from blood clots, washed in distilled water to remove gross debris, and finally rinsed in several changes of sterile saline. Some of the worms were frozen at -70 C and stored at -20 C; others were lyophilized before storage at -20 C. A homogenate of partially thawed D. immitis was prepared in PBS (phosphate-buffered saline 0.15 M, pH 7.2) at 5 C with a Waring blendor surrounded by CO2 ice. The amount of PBS used was 20 times the wet weight of the worms. The suspension of worm tissue was placed in a refrigerator (5 C), stirred slowly with a magnetic stirrer for approximately 16 hr, and then centrifuged (2,750 g, 30 min). Part of the supernatant fluid was used to prepare other fractions and the remainder was lyophilized and stored at -20 C. Whole worm PBS extract was mixed with an equal volume of cold (-20 C) 95% ethanol, left 4 hr in a refrigerator (5 C), and then centrifuged (2,750 g, 30 min). The precipitate was suspended in PBS and centrifuged (850 g, 30 min) to remove any insoluble particles; the supernatant fluid was lyophilized.