Dextran Density Gradient Purification of Plasmodium Sporozoites

Abstract

Several procedures have been described for isolating relatively pure preparations of Plasmodium sporozoites from homogenates of infected mosquitoes. These have included gradient centrifugation (on continuous or discontinuous diatrizoate gradients), column purification (with ion-exchange or lectin affinity columns), and filtration through pores of defined size (review and comparative assessment of these techniques in Vanderberg, 1980, In The in vitro cultivation of the pathogens of tropical diseases, W.H.O. Tropical Diseases Research Series, 3, Schwabe & Co., Basel, pp. 77-89). We now report a new gradient procedure, which yields better and more consistent results than any previously described. Plasmodium berghei sporozoites were obtained from Anopheles stephensi mosquitoes infected 18-22 days previously (procedures as in Vanderberg, 1977, Experimental Parasitology 42: 169-181). Generally, 3 separate batches of 100200 mosquitoes were gently homogenized for 2 min with 1.5 ml Medium M199 (GIBCO Labs., Grand Island, New York) in an ice-chilled mortar. The pooled homogenates were transferred to a 15-ml centrifuge tube, and additional medium added to make a total of 10 ml. The preparation was centrifuged at 20 g for 6 min, after which the supernatant suspension was transferred to a 12-ml glass Sorvall centrifuge tube. The pellet, containing residual sporozoites, was returned to a mortar and gently ground with 2 ml M199 for 2 min; the resulting homogenate was transferred to a 15-ml centrifuge tube, additional M199 to