Characterization of Antigens from Leishmania mexicana Grown in Dialysate Culture

Abstract

An extract of Leishmania mexicana grown in dialysate medium was prepared by sonication, dialysis, and lyophilization. Rabbits were immunized with the extract emulsified in Freund's incomplete adjuvant. Disc electrophoresis and immunoelectrophoresis procedures were performed on the extract. The results indicate that L. mexicana is highly complex in its antigenic make-up. Through disc electrophoresis of the extract, the presence of DNA, carbohydrate, and lipid, as well as at least 8 separate protein fractions, have been confirmed. Eleven antigens were shown to be present in the extract through immunoelectrophoresis. The dialysis culture technique was found to be the method of choice for the cultivation of leptomonads to be used in immunological studies. Little information is available on the antigenic make-up of the protozoan genus Leishmania. Leptomonad extracts obtained from several different species exhibited four to six precipitin bands when tested with homologous antisera in immunodiffusion studies reported by Bray and Lainson (1966). Schneider and Hertig (1966) demonstrated up to five precipitin bands by immunodiffusion. At least four precipitin bands were seen by Garcia (1965) when L. tropica was studied by immunodiffusion. One of the antigenic components was stable when heated to 60 C for 1 hr. Antigenic changes occurring in the leishmania-leptomonad transformation of L. donovani have recently been reported by Simpson (1968). The first objective of the present study was to evaluate the media available for culturing the leptomonad form in vitro, with particular emphasis on the value of these media in immunological studies. Aside from the desirable attribute of producing a large growth yield per unit volume, two other prerequisites were established for any medium or culture technique selected. The harvest should be relatively free from contaminating materials, such as red blood cells, and the medium should be easy to prepare and use in large quantities for batch culture. The final objective was to characterize an extract from leptomonad forms by disc electrophoretic and immunoelectropho-