Analysis of the Antigenic Relationships Among Trichomonas,Histomonas, Dientamoeba,and Entamoeba. I. Quantitative Fluorescent Antibody Methods*

SYNOPSIS. Antigens were prepared from axenic Entamoeba histolytica, Entamoeba invadens, and Trichomonas gallinae; dixenic Dientamoeba fragilis; and agnotobiotic Histomonas meleagridis cultures. Antisera were developed in rabbits against each of these species by subcutaneous inoculations of homogenized organisms with complete Freund's adjuvant. The globulin fraction of each serum was conjugated with fluorescein isothiocyanate (FITC) and then processed on Sephadex G-25 and DEAE-cellulose columns. Fluorescein/protein ratios were determined for the several DEAE fractions obtained from each of the 5 conjugated globulins, and those with ratios of approximately 3.0 were selected for use in all experiments. Conjugated anti-Dientamoeba and anti-Histomonas fractions were absorbed with the bacterial flora present in the respective cultures before being used for staining. Intact, formalin-fixed organisms of each of the species were subjected to direct staining, inhibition staining, and staining with cross-absorbed conjugated fractions. The emitted fluorescence was measured in an ultramicrofluorimeter. Cross reactions among the 5 antigens and 5 conjugated antisera suggested that very few, if any, common antigens were shared by Trichomonas and Entamoeba. They indicated also a close antigenic relationship between Trichomonas and Histomonas on the one hand and between Histomonas and Dientamoeba on the other. Trichomonas and Dientamoeba appeared to be less closely related, and still less relationship was noted between Dientamoeba and Entamoeba. Only very weak reactions were recorded between Histomonas and Entamoeba. Entamoeba invadens emitted much fluorescence after being stained with anti-Entamoeba histolytica conjugate and similar results were obtained by reciprocal staining. The phylogenetic implications of the immunologic findings are discussed.