Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro.
An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 x 10(4) human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid), 50 mM glucose, 1 microgram ml-1 folic acid, 4 micrograms ml-1 4-aminobenzoic acid, 2 micrograms ml-1 pantothenic acid and 35 micrograms ml-1 ascorbic acid. Incubation conditions were at 37 degrees C in a 5% CO2/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5-3.0 x 10(4) oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.
Woods
K M
KM
Division of Biology, Kansas State University, Manhattan 66506, USA.
Nesterenko
M V
MV
Upton
S J
SJ
D000818:Animals; D002478:Cells, Cultured; D003049:Coccidiostats / Q000494:pharmacology; D016785:Cryptosporidium parvum / Q000187:drug effects / Q000254:growth & development*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007106:Immune Sera; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D003049:Coccidiostats; D007106:Immune Sera