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Analysis‐only detection of Giardia by combining immunomagnetic separation and two‐color flow cytometry
Author(s) -
Ferrari Belinda C.,
Veal Duncan
Publication year - 2003
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.10009
Subject(s) - giardia , immunomagnetic separation , flow cytometry , monoclonal antibody , biology , microbiology and biotechnology , staining , immunofluorescence , giardia lamblia , primary and secondary antibodies , antibody , chromatography , chemistry , immunology , genetics
Background Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required. Methods Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre‐enrichment step was compared with filtration of the water sample. Cysts were recovered with two‐color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb‐FITC and mAb‐PE. Results A fourfold increase in fluorescent signal intensity was obtained when combining the mAb‐PE and PNA probe compared with two‐color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected. Conclusions Analysis‐only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary. Cytometry Part A 51A:79–86, 2003. © 2003 Wiley‐Liss, Inc.