Electrophoretic and immunoblot analysis of Cryptosporidium oocysts

Abstract

Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat-derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS-PAGE. Fifty-one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14 000–200 000 molecular weight (MW), two bands were less than 14 000 MW and one band was above 200 000 MW. Twenty-one bands were detected in the oocyst wall preparation, all within the range 14 000–200 000 MW. Immunoblot analysis of Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two serum groups. Nine of the 10 human sera and all of the goat kid sera reacted with a 23 000 MW and 32 000 MW antigen. A 15 500 MW antigen was also detected by all the goat and four of the 10 human sera. Both serum groups reacted with various antigens above 40 000 MW. Surface labelling of three human isolates of Cryptosporidium oocysts with ¹²⁵I was performed using the Bolton and Hunter reagent. The solubilized preparations were separated by SDS-PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15 500, 32 000, 47 500, 79 000 and 96 000 MW. Some variation in the molecular weight of polypeptides labelled with ¹²⁵I was observed among the three isolates. These studies indicate that the 32 000 MW antigen appears to be a suitable candidate for the preparation of an antibody probe because it is common to many isolates and is located on the exterior of the oocyst wall.