Precipitate Patterns in Agar Gel with Sera from Human Amebiasis and Entamoeba histolytica Antigen

Abstract

Immunodiffusion has been employed in studying antigen-antibody reactions related to Entamoeba histolytica in human infections. As many as ten arcs of precipitates have been demonstrated in sera from symptomatic infections occurring in man. Precipitins were observed in 16 confirmed cases and in none of 12 normal individuals. A generally available standardized antigen is needed to implement serological studies of amebiasis. Precipitins related to infections with Entamoeba histolytica were reported by Wagener (1924) in sera of cats with experimental amebic dysentery. More recently the utilization of agar gel as a medium for such reactions has been accompanied by increased interest in the study of precipitins generally. Maddison and Elsdon-Dew (1961) in certain cases of amebiasis in man noted the presence of nonspecific antibodies by means of a macrodiffusion procedure using agar gel. An improvement in the application of gel diffusion to analysis of antigen-antibody precipitates generally was achieved by Wadsworth (1957). This adaptation consisted of a miniature technique known as microdiffusion. It was described as more economical of time and material as well as possessing greater sensitivity. A scaled-down method of agar-gel diffusion was employed in the present study to investigate further the precipitins in sera from human instances of amebiasis. MATERIALS AND METHODS Cultures of E. histolytica (NIH 200 strain) for antigenic purposes were obtained by use of the medium of Cleveland and Collier. All cultures were incubated in an atmosphere of 5% carbon dioxide. Trophozoites from 48-hr cultures were pooled and washed four to six times by mixing with Locke's solution to give a tenfold dilution, followed by centrifuging at approximately 40 g. After the last washing the sediment was diluted to a volume of 5 ml with Locke's solution, and duplicate counts of amebas made by means of a hemocytometer. The material again was centrifuged and the sediment transferred to a small rubbercapped vial with from 1 to 5 ml of Locke's solution. Vials then were stored temporarily at -60 C. Further preparation of antigen consisted first in thawing the material so that the amebas were disintegrated. After centrifugation at 18,000 g for 45 min in a cold room at 4 C, the sediment was reslurried with one or two volumes of saline or water and again centrifuged. The supernatant was ultrafiltered through cellulose dialysis tubing of %A2-inch diameter under a vacuum of 5 to 10 cm Hg at 4 C. The extract was centrifuged once more for clarification and stored in a vial at -60 C. Antigenic material in 1 ml of the final product was derived from an estimated 84 million amebas. Since the cultures of E. histolytica included a mixed flora of bacteria, extracts of these organisms were prepared for controls in the tests. The bacterial associates were cultured in the same medium used for E. histolytica and subsequently disintegrated by sonic oscillation,1 20 kc/sec for 30 min. After centrifugation at 18,000 g, the supernatant was concentrated by ultrafiltration as explained above for the amebic preparation. Samples of human sera2 were collected from 16 cases of amebiasis occurring in African males. Eleven were confirmed instances of acute amebic dysentery, four were cases of liver abscess proved by aspiration of pus, and one individual was a cystpasser with peptic ulcer but without symptoms of amebiasis. Additional sera to serve as controls were acquired from 12 individuals in our own laboratory in whom Received for publication 25 January 1963. MSE-Mullard Ultrasonic Disintegrator, 60-watt model. The use of trade names is for identification only and does not constitute product-endorsement by the U. S. Public Health Service. 2 Sera were obtained through the courtesy of Drs. R. Elsdon-Dew and S. J. Powell, Amoebiasis Research Unit, Durban, South Africa.