Cryptosporidium merozoite isolation and purification using differential centrifugation techniques.
Simple modifications to a recently published merozoite purification procedure (Bjorneby et al., J. Immunol. 145:298, 1990) increased yields 3- to 5-fold. Calves were infected with 2.5 x 10(8) Cryptosporidium parvum oocysts and sacrificed 65 h post-infection. The ilium and caecum were removed. The tissue was sieved through a large strainer (2 mm2) to produce a homogeneous suspension. Red blood cells were removed by differential centrifugation (600 g); merozoites remained in the supernatant. The merozoites were pelleted (2,100 g) and washed in modified Hank's balanced salt solution deficient in Mg+2 and Ca+2. Percoll purification (density 1.070 g/ml and centrifugation speed of 22,000 g for 30 min) yielded 8 x 10(8) merozoites. Nineteen monoclonal antibodies (MAb) detected by either an enzyme-linked immunosorbent assay or an immunofluorescence assay, have been generated against the merozoite stage. Gels of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-stained showed that sporozoites and merozoites have many common lower molecular weight proteins. Western blots of sporozoite and merozoite antigens reacted with anti-sporozoite MAb showed several cross-reacting antigens shared by these life-cycle stages.
Regan
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Department of Veterinary Science, University of Arizona, Tucson 85721.
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Sterling
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D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D002499:Centrifugation, Density Gradient; D016785:Cryptosporidium parvum / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007421:Intestine, Small / Q000469:parasitology; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal