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 104 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 (JV‐2‐hydroxyethylpiperazine N−2‐ethanesulfonic acid), 50 mM glucose, 1 μg ml−1 folic acid, 4 μg ml−1 4‐aminobenzoic acid, 2 μg ml−1 pantothenic acid and 35 μg ml−1 ascorbic acid. Incubation conditions were at 37 ° 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 × 104 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.