Cryptosporidium merozoite isolation and purification using differential centrifugation techniques.

S. Regan, V. Cama, Charles R Sterling

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
JournalJournal of Protozoology
Volume38
Issue number6
StatePublished - Nov 1991

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Merozoites
Cryptosporidium
Centrifugation
Sporozoites
Monoclonal Antibodies
Ilium
Cryptosporidium parvum
Antigens
Oocysts
Life Cycle Stages
Silver
Sodium Dodecyl Sulfate
Fluorescent Antibody Technique
Polyacrylamide Gel Electrophoresis
Suspensions
Proteins
Salts
Erythrocytes
Molecular Weight
Western Blotting

ASJC Scopus subject areas

  • Parasitology

Cite this

Cryptosporidium merozoite isolation and purification using differential centrifugation techniques. / Regan, S.; Cama, V.; Sterling, Charles R.

In: Journal of Protozoology, Vol. 38, No. 6, 11.1991.

Research output: Contribution to journalArticle

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