title: Analysis-only detection of Giardia by combining immunomagnetic separation and two-color flow cytometry.
abstract: 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.
authors.affiliation: Centre for Fluorometric Applications in Biotechnology, School of Biological Sciences, Macquarie University, Sydney, Australia. bferrari@rna.bio.mq.edu.au
authors.forename: Belinda C
authors.initials: BC
authors.lastname: Ferrari
authors.forename: Duncan
authors.initials: D
authors.lastname: Veal
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D016829:Giardia lamblia / Q000166:cytology / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D015203:Reproducibility of Results
chemical_list: D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes