title: Standardization of a fluorescent-based quantitative adhesion assay to study attachment of Taenia solium oncosphere to epithelial cells in vitro.
abstract: To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment.
authors.affiliation: Laboratory of Infectious Diseases Research, Department of Cellular and Molecular Sciences, School of Sciences and Philosophy Universidad Peruana Cayetano Heredia, PO Box 5045, Lima, Peru.
authors.forename: Nancy
authors.initials: N
authors.lastname: Chile
authors.forename: Julio
authors.initials: J
authors.lastname: Evangelista
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.forename: Yanina
authors.initials: Y
authors.lastname: Arana
authors.forename: Sandra
authors.initials: S
authors.lastname: Palma
authors.forename: Charles R
authors.initials: CR
authors.lastname: Sterling
authors.forename: Hector H
authors.initials: HH
authors.lastname: Garcia
authors.forename: Armando
authors.initials: A
authors.lastname: Gonzalez
authors.forename: Manuela
authors.initials: M
authors.lastname: Verastegui
mesh_terms: D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002448:Cell Adhesion / Q000276:immunology*; D006224:Cricetinae; D004847:Epithelial Cells / Q000276:immunology* / Q000469:parasitology; D005455:Fluorescent Antibody Technique / Q000379:methods*; D019073:HT29 Cells; D006801:Humans; D041201:Taenia solium / Q000276:immunology*