title: Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.
abstract: Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-μm filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 μm) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, INTA-CSIC).
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Blanco
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, INTA-CSIC).
authors.forename: Mercedes
authors.initials: M
authors.lastname: Moreno-Paz
authors.affiliation: Department of Molecular Evolution, Centro de Astrobiología (CAB, INTA-CSIC); parrogv@cab.inta-csic.es.
authors.forename: Victor
authors.initials: V
authors.lastname: Parro
mesh_terms: D000907:Antibodies, Bacterial / Q000032:analysis*; D000458:Cyanobacteria / Q000276:immunology / Q000302:isolation & purification*; D017753:Ecosystem; D004784:Environmental Monitoring / Q000379:methods*; D005068:Eutrophication; D005618:Fresh Water / Q000382:microbiology*; D046228:Microarray Analysis / Q000379:methods*; D015203:Reproducibility of Results; D014871:Water Microbiology
chemical_list: D000907:Antibodies, Bacterial