Standardization of a fluorescent-based quantitative adhesion assay to study attachment of Taenia solium oncosphere to epithelial cells in vitro

Nancy Chile, Julio Evangelista, Robert H. Gilman, Yanina Arana, Sandra Palma, Charles R. Sterling, Hector H. Garcia, Armando Emiliano Gonzalez Zariquiey, Manuela Verastegui

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)89-96
Number of pages8
JournalJournal of Immunological Methods
Volume376
Issue number1-2
DOIs
StatePublished - 28 Feb 2012
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by The Fogarty International Clinical Research Scholars and Fellows Program at Vanderbilt University ( R24 TW007988 ), Bill and Melinda Gates Foundation — Cysticercosis Elimination in Peru (grants 23981 and 33848 ), 5D43TW006581 National Institutes of Health “Infectious Diseases Training Program in Peru”, Training Grant ( FIC/NIH D43 TW001140 ) and the RG-ER anonymous fund for Tropical Research. We thank Dr. Karen Alroy for revising and editing the manuscript, Dr. Eric Deharo and Dr. Yanick Estevez for help with the use of the Chamaleon V Microplate Reader, and the technical assistance of J.B. Phu, D. and Sara.

Keywords

  • Biotinylation
  • Epithelial cells
  • Quantitative adhesion assay
  • Taenia solium oncosphere

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