Antigenic Analysis of Entamoeba histolytica by Means of Fluorescent Antibody. IV. Relationships of Two Strains of E. histolytica and One of E. hartmanni Demonstrated by Cross-Absorption Techniques

Abstract

Antiserum was prepared against E. histolytica K9, E. histolytica Huff, and E. hartmanni 335. After being tagged with fluorescein, portions of each antiserum were treated with large numbers of homologous and heterologous amebae in order to absorb out specific antibodies. The resulting treated antisera were then reacted with each of the ameba strains under conditions that permitted quantitative evaluation of the staining results. These experiments demonstrated that the two E. histolytica strains did not remove specific antibody from E. hartmanni antisera, although they did remove specific antibody from E. histolytica antisera. The converse was also true. Finally, there was evidence that E. histolytica K9 shared some antigens with E. histolytica Huff, but possessed others which were not shared. In a previous paper of this series (Goldman et al., 1960) it was shown that E. histolytica and E. hartmanni were readily distinguishable from each other on the basis of the degree to which each reacted with homologous and heterologous antisera. In addition, there were indications of antigenic differences between two strains of E. histolytica. The present paper reports a more detailed investigation of these relationships based upon cross-absorption of antibodies by means of large numbers of intact amebae. Although this technique for selective absorption of antibodies is widely practiced in studying antigenic relationships of other microorganisms, to our knowledge the work described here represents the first application of the method to the intestinal amebae of man. MATERIALS AND METHODS Strains of amebae: E. histolytica K9 (E. h. K9), virulent for man; E. histolytica Huff (E. h. Huff), avirulent for man; and E. hartmanni 335 (E. hart. 335) were studied. The origins of these strains and details of culture maintenance have been described previously (ibid.). Preparation of antisera: Two young adult rabbits were used for each strain. Before immunization all six rabbits were bled and portions from each bleeding were pooled to form the normal rabbit serum control for this study. Similar immunization schedules were employed for both E. h. K9 and E. hart. 335. With each of these strains about Received for publication 11 May 1962. *Present address: Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. 15 million lyophilized organisms, suspended in Freund's adjuvant, were inoculated subcutaneously into each rabbit at each of three intervals, 14 days apart. Four weeks later a booster inoculation of 11 million organisms was administered, and 11 weeks later another booster was given. In all about 60 million amebae were administered to each rabbit, the animals being bled at intervals during this immunization period. The antisera that were labeled with fluorescein were drawn 70 days after the ini ial inoculation in the case of E. h. K9, and 143 days in the case of E. hart. 335. The schedule of E. h. Huff was more varied. The first three inoculations consisted of a total of 45 million lyophilized amebae suspended in Freund's adjuvant, aluminum hydroxide adjuvant and distilled water respectively. They were given 1 week apart, the first two subcutaneously, the third intravenously. Booster inoculations were administered either intravenously or subcutaneously at intervals of 3, 6, and 8 weeks later. Eight days later the rabbits were bled. In all, about 65 million amebae were administered, and the final bleeding took place 98 days after the initial inoculation. All sera, including the normal pool, were fractionated with ammonium sulfate and the crude globulin portions were labeled with fluorescein isothiocyanate by the methods generally followed in this laboratory (Cherry et al., 1960). To reduce nonspecific staining the conjugates were exposed three times to washed hog liver powder at the rate of 100, 80, and 60 mg of powder per ml of conjugate, respectively. By centrifuging the conjugates in a Hemming centrifugal filter assembly fitted with filter paper instead of a sterilizing pad, about 95% of the original volume was recovered after three treatments. All four conjugates were adjusted to the same level of fluorescence by dilution with 2.5% bovine serum albumin (BSA) in veronal buffer, pH 7.5. One-step inhibition tests (Goldman, 1957) with each conjugate and its parent serum showed distinct blocking activity in every