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<article dtd-version="3.0" article-type="Invited Methods Article - JoVE Produced Video">
  <front>
    <journal-meta>
      <journal-id journal-id-type="publisher">jove</journal-id>
      <journal-id journal-id-type="email">editorial-office@jove.com</journal-id>
      <journal-title-group>
        <journal-title>Journal of Visualized Experiments</journal-title>
      </journal-title-group>
      <issn pub-type="ppub">1940-087X</issn>
      <issn pub-type="epub"></issn>
    </journal-meta>
    <article-meta>
      <article-id pub-id-type="publisher-id">JoVE54994</article-id>
      <article-id pub-id-type="manuscript">JoVE54994</article-id>
      <article-categories>
        <subj-group subj-group-type="Article Type">
          <subject>Invited Methods Article - JoVE Produced Video</subject>
        </subj-group>
        <subj-group subj-group-type="Category">
          <subject>JoVE Environment</subject>
        </subj-group>
        <subj-group subj-group-type="Classification">
          <subject>Microbiology</subject>
          <subject>Environmental Microbiology</subject>
        </subj-group>
      </article-categories>
      <title-group>
        <article-title>Experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip</article-title>
      </title-group>
      <contrib-group>
        <contrib contrib-type="author">
          <role content-type="1" />
          <name>
            <surname>Blanco</surname>
            <given-names>Yolanda</given-names>
          </name>
          <email>blancoly@cab.inta-csic.es</email>
          <xref ref-type="aff" rid="aff1" />
        </contrib>
        <contrib contrib-type="author">
          <role content-type="2" />
          <name>
            <surname>Moreno-Paz</surname>
            <given-names>Mercedes</given-names>
          </name>
          <email>morenopm@cab.inta-csic.es</email>
          <xref ref-type="aff" rid="aff2" />
        </contrib>
        <contrib contrib-type="author" corresp="yes">
          <role content-type="3" />
          <name>
            <surname>Parro</surname>
            <given-names>Victor</given-names>
            <prefix>Dr.</prefix>
          </name>
          <email>parrogv@cab.inta-csic.es</email>
          <xref ref-type="aff" rid="aff3" />
        </contrib>
        <contrib contrib-type="editor">
          <name>
            <surname>Nguyen</surname>
            <given-names>Nam</given-names>
          </name>
          <degrees>Ph.D.</degrees>
          <email>nam.nguyen@jove.com</email>
          <xref ref-type="aff" rid="aff4" />
        </contrib>
        <aff id="aff1">
          <institution>Centro de Astrobiologia (INTA-CSIC)</institution>
          <institution content-type="dept">Molecular Evolution</institution>
          <addr-line content-type="addrline1">Carretera de Ajalvir km 4</addr-line>
          <country>SPAIN</country>
          <addr-line content-type="city">Torrejón de Ardoz, Madrid</addr-line>
          <addr-line content-type="state">Madrid</addr-line>
          <addr-line content-type="zipcode">28850</addr-line>
          <institution>Department of Molecular Evolution
Centro de Astrobiología (CAB, INTA-CSIC), Carretera de Ajalvir km 4
Torrejón de Ardoz, 28850, Madrid, Spain.</institution>
        </aff>
        <aff id="aff2">
          <institution>Centro de Astrobiologia (INTA-CSIC)</institution>
          <institution content-type="dept">Molecular Evolution</institution>
          <addr-line content-type="addrline1">Carretera de Ajalvir km 4</addr-line>
          <country>SPAIN</country>
          <addr-line content-type="city">Torrejón de Ardoz, Madrid</addr-line>
          <addr-line content-type="state">Madrid</addr-line>
          <addr-line content-type="zipcode">28850</addr-line>
        </aff>
        <aff id="aff3">
          <institution>Centro de Astrobiologia (INTA-CSIC)</institution>
          <institution content-type="position">Research Scientist</institution>
          <institution content-type="dept">Molecular Evolution</institution>
          <addr-line content-type="addrline1">Carretera de Ajalvir km 4</addr-line>
          <country>SPAIN</country>
          <addr-line content-type="city">Torrejón de Ardoz</addr-line>
          <addr-line content-type="state">Madrid</addr-line>
          <addr-line content-type="zipcode">28850</addr-line>
          <fax>+34-915201074</fax>
          <phone content-type="primary">+34-915201071</phone>
          <institution>Centro de Astrobiologia (INTA-CSIC)</institution>
        </aff>
        <aff id="aff4">
          <institution>JoVE, Journal of Visualized Experiments</institution>
          <addr-line>1 Alewife Center</addr-line>
          <addr-line>Suite 200</addr-line>
          <addr-line>Cambridge, MA  02140</addr-line>
          <country>UNITED STATES</country>
        </aff>
      </contrib-group>
      <history>
        <date date-type="received">
          <day>12</day>
          <month>05</month>
          <year>2016</year>
        </date>
        <date date-type="rev-recd">
          <day>19</day>
          <month>09</month>
          <year>2016</year>
        </date>
        <date date-type="rev-recd">
          <day>10</day>
          <month>06</month>
          <year>2016</year>
        </date>
        <date date-type="accepted">
          <day>22</day>
          <month>09</month>
          <year>2016</year>
        </date>
      </history>
      <abstract>
        <p>Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth with relevant health and economic consequences. Many cyanobacterial strains are toxin producers and few cells are necessary to induce irreparable damages to the environment. Therefore, water body authorities and administration require rapid and efficient early warning systems providing reliable data for supporting their preventive or curative decisions. This manuscript reports an experimental protocol for in field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Herein, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for simultaneous monitoring of 17 cyanobacterial strains frequently found in blooming in freshwater ecosystems, some of them toxin producers is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP onto a single microscope slide was printed to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the Abs or after cell concentration either by filtration through 1-3 μm filter. Solid samples as sediments and ground rocks are firstly homogenized and dispersed by a hand-held ultrasonicator into an incubation buffer, filtered (5-20 μm) to remove the course material, and the filtrate incubated with the Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence -labelled Abs and read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to 2 1-hr periods of incubation. The output is an image where bright spots correspond to the positive detection of cyanobacterial markers.</p>
      </abstract>
      <kwd-group>
        <kwd>Cyanobacteria, CYANOCHIP, environmental monitoring, antibody microarray, portable fluorescent immunoassay, freshwater reservoir monitoring</kwd>
      </kwd-group>
      <funding-group />
      <counts>
        <fig-count count="0" />
      </counts>
      <custom-meta-group>
        <custom-meta>
          <meta-name>Please indicate whether this article will be standard or open access. </meta-name>
          <meta-value>Standard Access (US$2,400);</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Please indicate the &lt;b&gt; full address&lt;/b&gt; at which this article will be &lt;b&gt;filmed&lt;/b&gt;.</meta-name>
          <meta-value>Centro de Astrobiología (INTA-CSIC), Carretera de Ajalvir km4, Torreón de Ardoz, 28850, Madrid, Spain</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Is all of the written text in your manuscript original (not plagiarized, or self-plagiarized)? If using previously published figures, are re-use permissions from the previous publishers obtained and cited in the figure legends?</meta-name>
          <meta-value>Yes</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Black and White Image Count</meta-name>
          <meta-value>0</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Color Image Count</meta-name>
          <meta-value>0</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Acquisitions Editor</meta-name>
          <meta-value>Alison Hamlin</meta-value>
        </custom-meta>
        <custom-meta>
          <meta-name>Assistant who entered lead</meta-name>
          <meta-value>Risa McNellis</meta-value>
        </custom-meta>
      </custom-meta-group>
    </article-meta>
  </front>
  <body>
    <fig>
      <label>Figure</label>
      <graphic xlink:href="Figure1.tif" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <table-wrap>
      <label>Table</label>
      <graphic xlink:href="Table 1.docx" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <table-wrap>
      <label>Excel Spreadsheet- Table of Materials/Equipment</label>
      <graphic xlink:href="BlancoEtal_JoVE Materials.xls" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <table-wrap>
      <label>Excel Spreadsheet- Table of Materials/Equipment</label>
      <graphic xlink:href="JoVE_Materials_Blanco et al.xls" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
    <fig>
      <label>Figure1</label>
      <graphic xlink:href="Diapositiva1.tiff" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </fig>
    <table-wrap>
      <label>Table1</label>
      <graphic xlink:href="Table1.xlsx" xmlns:xlink="http://www.w3.org/1999/xlink" />
    </table-wrap>
  </body>
</article>