Name: experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip
Uploaded: 2017-02-07T12:30:00
Description: Watch this Scientific Journal Video about Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip at JoVE.com

We thank Dr. Antonio Quesada from the Universidad Aut&#243;noma de Madrid for providing cyanobacterial strains. This work was funded by the Subdirecci&#243;n General de Proyectos de Investigaci&#243;n of the Spanish Ministerio de Econom&#237;a y Competitividad (MINECO), grants no. AYA2011-24803 and ESP2014-58494-R.

1. Preparation of the Immunogens
<ol>
	<li>Grow each cyanobacterial strain in the corresponding culture medium under conditions described in Table 1. 
	NOTE: Growth medium and culture conditions for each cyanobacteria strain are listed in Table 1. All the cyanobacterial strains, with the exception of K17, belong to Antonio Quesada&#39;s group from Autonoma University (Madrid, Spain). The antibody against Planktothrix rubescens was generated from a natural sample ...

<ol>
	<li>Simis, S. G. H., Peters, S. W. M., Gons, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+sensing+of+the+cyanobacterial+pigment+phycocianin+in+turbid+inland+water.">Remote sensing of the cyanobacterial pigment phycocianin in turbid inland water.</a> Limnol. Oceanogr. 50 (1), 237-245 (2005).</li><li>Zamyadi, A., McQuaid, N., Prevost, M., Dorner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+potentially+toxic+cyanobacteria+using+an+online+multi-probe+in+drinking+water+sources.">Monitoring of potentially toxic cyanobacteria using an online multi-probe in drinking water sources.</a> J. Environ. Monit. 14, 579-588 (2012).</li><li>Msagati, T. A. M., Siame, B. A., Shushu, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+the+isolation,+detection+and+quantification+of+cyanobacterial+hepatotoxins.">Evaluation of methods for the isolation, detection and quantification of cyanobacterial hepatotoxins.</a> Aquat. Toxicol. 78 (4), 382-397 (2006).</li><li>Weller, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoassays+and+biosensors+for+the+detection+of+cyanobacterial+toxins+in+water.">Immunoassays and biosensors for the detection of cyanobacterial toxins in water.</a> Sensors. 13 (11), 15085-15112 (2013).</li><li>Baker, J. A., Neilan, B. A., Entsch, B., McKay, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cyanobacteria+and+their+toxigenicity+in+environmental+samples+by+rapid+molecular+analysis.">Identification of cyanobacteria and their toxigenicity in environmental samples by rapid molecular analysis.</a> Environ. Toxicol. 16 (6), 472-482 (2001).</li><li>Li, H., Singh, A. K., McIntyre, L. M., Sherman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+in+response+to+hydrogen+peroxide+and+the+putative+PerR+regulon+of+Synechocystis+sp.+Strain+PCC+6803.">Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp. Strain PCC 6803.</a> J. Bacteriol. 186 (11), 3331-3345 (2004).</li><li>Anderson, D. M., Cembella, A. D., Hallegraeff, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+understanding+harmful+algal+blooms:+paradigm+shifts+and+new+technologies+for+research,+monitoring,+and+management.">Progress in understanding harmful algal blooms: paradigm shifts and new technologies for research, monitoring, and management.</a> Ann. Rev. Mar. Sci. 4, 143-176 (2012).</li><li>Dittami, S. M., Pazos, Y., Laspra, M., Medlin, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+testing+for+the+presence+of+toxic+algae+monitoring+programme+in+Galicia+(NW+Spain).">Microarray testing for the presence of toxic algae monitoring programme in Galicia (NW Spain).</a> Environ. Sci. Pollut. Res. Int. 20, 6778-6793 (2013).</li><li>Kegel, J. U., Del Amo, ., Medlin, Y., K, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Project+MIDTAL:+its+methods+and+samples+from+Arcachon+Bay,+France.">Introduction to Project MIDTAL: its methods and samples from Arcachon Bay, France.</a> Environ. Sci. Pollut. Res. Int. 20 (10), 6690-6704 (2013).</li><li>Marcheggiani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+emerging+and+re-emerging+pathogens+in+surface+waters+close+to+an+urban+area.">Detection of emerging and re-emerging pathogens in surface waters close to an urban area.</a> Int. J. Environ. Res. Public Health. 12 (5), 5505-5527 (2015).</li><li>Fern&#225;ndez-Calvo, P., N&#228;ke, C., Rivas, L. A., Garc&#237;a-Villadangos, M., G&#243;mez-Elvira, J., Parro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multi-array+competitive+immunoassay+for+the+detection+of+broad-range+molecular+size+organic+compounds+relevant+for+astrobiology.">A multi-array competitive immunoassay for the detection of broad-range molecular size organic compounds relevant for astrobiology.</a> Planet. Space Sci. 54 (815), 1612-1621 (2006).</li><li>Parro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Antibody microarrays for environmental monitoring. Handbook of Hydrocarbon and Lipid Microbiology. , 2699-2710 (2010).</li><li>Fan, Z., Keum, Y. S., Li, Q. X., Shelver, W. L., Guo, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+immunoassay+detection+of+multiple+environmental+chemicals+on+protein+microarrays+using+DNA/dye+conjugate+as+a+fluorescent+label.">Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.</a> J. Environ. Monit. 14 (5), 1345-1352 (2012).</li><li>Rivas, L. A., Garc&#237;a-Villadangos, M., Moreno-Paz, M., Patricia Cruz-Gil, P., G&#243;mez-Elvira, J., Parro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+200-Antibody+microarray+biochip+for+environmental+monitoring:+Searching+for+universal+microbial+biomarkers+through+immunoprofiling.">A 200-Antibody microarray biochip for environmental monitoring: Searching for universal microbial biomarkers through immunoprofiling.</a> Anal. Chem. 80 (21), 7970-7979 (2008).</li><li>Delehanty, J. B., Ligler, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microarray+immunoassay+for+simultaneous+detection+of+proteins+and+bacteria.">A microarray immunoassay for simultaneous detection of proteins and bacteria.</a> Anal. Chem. 74 (21), 5681-5687 (2002).</li><li>Ligler, F. S., Taitt, C. R., Shriver-Lake, L. C., Sapsford, K. E., Shubin, Y., Golden, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Array+biosensor+for+detection+of+toxins.">Array biosensor for detection of toxins.</a> Anal. Bioanal. Chem. 377 (3), 469-477 (2003).</li><li>Rucker, V. C., Havenstrite, K. L., Herr, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+microarrays+for+native+toxin+detection.">Antibody microarrays for native toxin detection.</a> Anal. Biochem. 339 (2), 262-270 (2005).</li><li>Kang, J., Kim, S., Kwon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-based+biosensors+for+environmental+monitoring.">Antibody-based biosensors for environmental monitoring.</a> Toxicol. Environ. Health. Sci. 1 (3), 145-150 (2009).</li><li>Herranz, S., Marazuela, M. D., Moreno-Bondi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+portable+array+biosensor+for+multisample+microcystin+analysis+in+freshwater+samples.">Automated portable array biosensor for multisample microcystin analysis in freshwater samples.</a> Biosens. Bioelectron. 33 (1), 50-55 (2012).</li><li>Shlyapnikov, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+simultaneous+ultrasensitive+immunodetection+of+five+bacterial+toxins.">Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins.</a> Anal. Chem. 84 (13), 5596-5603 (2012).</li><li>Szkola, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+simultaneous+detection+of+ricin,+staphylococcal+enterotoxin+B+and+saxitoxin+by+chemiluminescence-based+microarray+immunoassay.">Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay.</a> Analyst. 139, 5885-5892 (2014).</li><li>Blanco, Y., Quesada, A., Gallardo-Carre&#241;o, I., Jacobo Aguirre, ., Parro, J., V, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CYANOCHIP:+An+antibody+microarray+for+high-taxonomical-resolution+cyanobacterial+monitoring.">CYANOCHIP: An antibody microarray for high-taxonomical-resolution cyanobacterial monitoring.</a> Environ. Sci. Technol. 49 (3), 1611-1620 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+Sensitive+Method+for+the+Quantitation+of+Microgram+Quantities+of+Protein+Utilizing+the+Principle+of+Protein-Dye+Binding.">A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.</a> Anal. Chem. 72, 248-254 (1976).</li><li>Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+measurement+with+the+Folin+phenol+reagent.">Protein measurement with the Folin phenol reagent.</a> J. Biol. Chem. 193 (1), 265-275 (1951).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+Bicinchoninic+acid.">Measurement of protein using Bicinchoninic acid.</a> Anal. Biochem. 150 (1), 76-85 (1985).</li><li>Rivas, L. A., Aguirre, J., Blanco, Y., Gonz&#225;lez-Toril, E., Parro, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graph-based+deconvolution+analysis+of+multiplex+sandwich+microarray+immunoassays:+applications+for+environmental+monitoring.">Graph-based deconvolution analysis of multiplex sandwich microarray immunoassays: applications for environmental monitoring.</a> Environ. Microbiol. 13 (6), 1421-1432 (2011).</li><li>Blanco, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+prokaryotic+community+and+metabolisms+in+south+african+deep-mine+biofilms+through+antibody+microarrays+and+graph+theory.">Deciphering the prokaryotic community and metabolisms in south african deep-mine biofilms through antibody microarrays and graph theory.</a> PloS One. 9 (2), e114180 (2014).</li><li>Gas, F., Baus, B., Quer&#233;, J., Chapelle, A., Dreanno, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+quantification+of+the+marine+toxic+algae,+Alexandrium+minutum,+using+a+super-paramagnetic+immunochromatographic+strip+test.">Rapid detection and quantification of the marine toxic algae, Alexandrium minutum, using a super-paramagnetic immunochromatographic strip test.</a> Talanta. 147, 581-589 (2016).</li><li>Parro, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOLID3:+a+multiplex+antibody+microarray-based+optical+sensor+instrument+for+in+situ+life+detection+in+planetary+exploration.">SOLID3: a multiplex antibody microarray-based optical sensor instrument for in situ life detection in planetary exploration.</a> Astrobiology. 11, 15-28 (2011).</li><li>McKay, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Icebreaker+Life+Mission+to+Mars:+a+search+for+biomolecular+evidence+for+life.">The Icebreaker Life Mission to Mars: a search for biomolecular evidence for life.</a> Astrobiology. 13, 334-353 (2013).</li></ol>

Here, a multiplex fluorescent sandwich immunoassay using the CYANOCHIP, a 17-antibody microarray for the detection and identification of a wide range of cyanobacterial genera, is described22. These cyanobacteria represent the most frequent benthic and planktonic genera in freshwater habitats, some of them being toxin producers. Recently, the fluorescent sandwich immunoassay format has been used to identify microorganisms and/or bioanalytes in environmental applications26...

The detection and monitoring of microorganisms in complex natural microbial communities are crucial in many fields, including biomedicine, environmental ecology, and astrobiology. Cyanobacteria are prokaryotic microorganisms well-known for their ability to form blooms (excessive proliferation) of cells in fresh water. They are ubiquitous, and many species are able to produce toxins, leading not only to a potential risk for human health, but also to an ecological impact. In this regard, it is essential to develop rapid and sensitive methods for the early detection of cyanobacteria and/or their toxins in soil and water. For this purpose, a multiplex fluorescent sandwich microarray immunoassay (FSMI) has been developed as a tool for water managers to help them in making decisions and, consequently, in implementing proper water management programs.
A diverse range of methods has been developed to detect and identify cyanobacterial cells and cyanotoxins in soil and water, including optical microscopy, molecular biology, and immunological techniques. These methods can vary greatly in the information they provide. Microscopic techniques are based on cell morphology and the detection of in vivo fluorescence from cyanobacterial pigments, such as phycocyanin or chlorophyll a1. Although they are quick and cheap methods for real-time and frequent monitoring that inform about the type and number of cyanobacteria present in a sample, they do not give information about the potential toxicity. In addition, they require a certain level of expertise, considering that it is often very difficult to distinguish between closely related species2. To overcome these limitations, light microscopy must be accompanied by both biological and biochemical screening assays and physicochemical methods for the identification and quantification of cyanotoxins.
Enzyme-linked immunosorbent assays (ELISA), protein phosphate inhibition assays (PPIA), and neurochemical tests in mice are examples of biochemical screening assays for the detection of cyanotoxins. While the first two are rapid and sensitive methodologies, false positives have been described when using ELISA and PPIA tests are restricted to three types of toxins. The mouse bioassay is a qualitative technique with low sensitivity and precision, and special licensing and training is required. In addition, it does not give information about the type of toxins present in a sample. Cyanotoxins can be identified and quantified by other analytical methods, such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), or matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). However, this is only possible if reference standards, which are needed to determine individual toxin concentrations in complex samples, are available3,4. Moreover, these methods are time-consuming; require costly equipment, supplies, and sample preparation; and must be performed by experienced and specialized staff.
Molecular-based methods have been applied for decades to detect, identify, and quantify cyanobacteria and their corresponding cyanotoxins thanks to the sequence information published in the genome databases (e.g., National Center for Biotechnology Information, NCBI). Among these methods are those based on the polymerase chain reaction (PCR), which requires the design of sets of primers for DNA amplification and depend on previous knowledge of DNA sequences of different cyanobacterial species. While gene detection, like the phycocyanin operon, leads to accurate identification at the genus level, some species or strains are undetected with this method. However, toxin-encoding genes, such as those belonging to the microcystin operon, facilitate the identification of toxins in samples where the producers are scarce5. Nonetheless, the detection of toxin markers by PCR does not necessarily imply toxicity in the environment. Furthermore, the set of primers developed to analyze the whole range of species of cyanobacteria and toxin producers present in a sample is still incomplete, and further studies must be done to identify unknown species. Other molecular techniques are non-PCR-based, such as fluorescence in situ hybridization (FISH) and DNA microarrays.
In the last two decades, microarray technology has gained importance in many fields of application, particularly in environmental monitoring. DNA microarrays allow for discrimination between species and analytes4,6,7,8,9,10, but they are considered very laborious and time-consuming tasks that involve multiple steps (e.g., microarray performance, DNA extraction, PCR amplification, and hybridization). For that reason, less time-consuming assays based on antibodies, such as sandwich and competitive immunological microarrays, have become an essential and reliable high-throughput method for the detection of multiple environmental analytes11,12,13. The capability of antibodies to specifically recognize their target compounds and to detect small amounts of analytes and proteins, along with the possibility of producing antibodies against almost any substance, make antibody microarrays a powerful technique for environmental purposes. In addition, the capability of achieving multiple analyses in a single assay, with limits of detection ranging from ppb to ppm, is one of the main advantages of this method14.
Antibody-based biosensors have proved to be sensitive and rapid tools for the detection of a wide range of pathogens and toxins in environmental monitoring15,16,17,18,19,20,21. While DNA methods involve several steps, the antibody-based microarrays only require a small sample preparation that is mainly based on a short lysis step in an appropriate solution buffer. Delehanty and Ligler15 reported the simultaneous detection of proteins and bacterial analytes in complex mixtures based on an antibody sandwich immunoassay capable of detecting a protein concentration of 4 ppb and 104 cfu/mL of cells. Szkola et al.21 have developed a cheap and reliable multiplex microarray for the simultaneous detection of proteotoxins and small toxins, compounds that might be used in biological warfare. They detected concentrations of ricin toxin, with a limit of detection of 3 ppb, in less than 20 min. Recently, the CYANOCHIP, an antibody microarray-based biosensor for the in situ detection of toxic and nontoxic cyanobacteria, has been described22. This microarray allows for the identification of potential cyanobacterial blooms, mostly in aquatic environments, which are difficult to identify microscopically. The limit of detection of the microarray is 102 - 103 cells for most species, turning this biosensor into a cost-effective tool for the multiplex detection and identification of cyanobacteria, even at the species level. All these properties make the antibody microarray technique, and particularly the method presented in this work, a quicker and simpler method compared to the aforementioned techniques.
This work presents two examples of experiments that use an antibody microarray-based biosensor to detect the presence of cyanobacteria in soil and water samples. It is a simple and reliable method based on a sandwich immunoassay format that requires very small sample volumes and very basic sample preparation. The method requires a short time and can be easily performed in the field.

<table border="0" cellpadding="0" cellspacing="0" width="1143">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">0.22 mm pore diameter filters</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">GSWP04700</td>
			<td style="width:427px;">For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Eppendorf&#160; 5424R microcentrifuge</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Phosphate buffer saline (PBS) pH 7.4 (10X)</td>
			<td style="width:248px;">Thermofisher Scientific</td>
			<td style="width:119px;">70011036</td>
			<td>50 mM potassium phosphate, 150 mM NaCl, pH 7.4</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Ultrasonic processor UP50H</td>
			<td style="width:248px;">Hielscher</td>
			<td style="width:119px;"></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Complete Freund&#39;s adjuvant&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">F5881</td>
			<td>Immunopotentiator</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Incomplete Freud&#39;s adjuvant</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">F5506</td>
			<td>For boost injections</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein A antibody purification kit&#160;&#160;</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">PURE1A&#160;</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Centrifugal filter devices MWCO&#60;100 KDa</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">UFC510096-96K</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Dialysis tubings, benzoylated</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">D7884-10FT</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">GE-HealthCare</td>
			<td style="width:119px;">GE27-5330-02</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Bradford reagent</td>
			<td style="width:248px;">Sigma-Aldrich</td>
			<td style="width:119px;">B6916-500 mL</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MicroBCA protein assay kit&#160;</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;">23235</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protein arraying buffer 2X</td>
			<td style="width:248px;">Whatman (Sigma Aldrich)&#160;</td>
			<td style="width:119px;">S00537</td>
			<td>Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Tween 20&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P9416</td>
			<td>Non-ionic detergent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Bovine serum albumin (BSA)</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">A9418</td>
			<td>Control&#160; for printing; blocking reagent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">384-wells microplate</td>
			<td style="width:248px;">Genetix</td>
			<td style="width:119px;">X6004</td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Robot arrayer for multiple slides</td>
			<td style="width:248px;">MicroGrid II TAS arrayer from Digilab</td>
			<td style="width:119px;"></td>
			<td>For antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Epoxy substrate glass slides</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td style="width:119px;">VEPO25C</td>
			<td>Solid support for antibody printing</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Alexa Fluor-647 Succinimidyl-ester&#160;</td>
			<td style="width:248px;">Molecular probes</td>
			<td style="width:119px;">A20006</td>
			<td>Fluorochrome</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">DMSO&#160;</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">D8418</td>
			<td>Fluorochrome dissolvent</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Titramax vibrating platform shaker</td>
			<td style="width:248px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For antibody labeling&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Illustra Microspin G-50 columns&#160;</td>
			<td style="width:248px;">Healthcare</td>
			<td style="width:119px;">27-5330-01</td>
			<td>For purification of labeled antibodies</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Safe seal brown 0,5 ml tubes</td>
			<td style="width:248px;">Sarstedt</td>
			<td style="width:119px;">72,704,001</td>
			<td>For labeled antibodies storage&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Nanodrop 1000 spectrophotometer</td>
			<td style="width:248px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td>To quantify antibody concentration and labeling efficiency</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">3 &#181;m pore size polycarbonate 47 mm diameter filter</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">TMTP04700</td>
			<td>To concentrate cells</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 8</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T3038</td>
			<td>For TBSTRR preparation; to block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Sodium chloride</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">S7653</td>
			<td>For TBSTRR preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">20 &#181;m nylon filters&#160;</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">NY2004700</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">10-12 mm filter holders</td>
			<td style="width:248px;">Millipore</td>
			<td style="width:119px;">SX0001300</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">P8340</td>
			<td>For environmental extract storage</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">1M Trizma hydrochloride solution pH 9</td>
			<td style="width:248px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T2819</td>
			<td>To block slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Heidolph Duomax 1030 rocking platform shaker</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;"></td>
			<td>To block slides; for incubation processes&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">VWR Galaxy miniarray microcentrifuge</td>
			<td style="width:248px;">VWR</td>
			<td style="width:119px;">C1403-VWR</td>
			<td>To dry slides</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Multi-Well microarray hybridization cassette</td>
			<td style="width:248px;">Arrayit corporation</td>
			<td>AHC1X24</td>
			<td>Cassette for 24 assays per slide</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix 4100A microarray scanner&#160;</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Scanner for fluorescence</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">GenePix Pro Software</td>
			<td style="width:248px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td>Software for image analysis and quantification</td>
		</tr>
	</tbody>
</table>

experimental protocol for detecting cyanobacteria in liquid and solid samples with an antibody microarray chip

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-&#956;m 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 &#956;m) 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.

This work describes a multiplex immunoassay test for the simultaneous identification of the most relevant freshwater cyanobacterial species (Table 1) using the CYANOCHIP antibody microarray. The microarray can be a 3 x 8 microarray format printed onto microscope slides. Each microarray is made up of a set of 17 antibodies printed in a triplicate spot pattern, their corresponding pre-immune antibodies and BSA as negative controls. The microarrays also include a fluorescent...

Watch this Scientific Journal Video about Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip at JoVE.com

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Research

JoVE Journal

Environment

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

Preparation and Testing of Impedance-based Fluidic Biochips with RTgill-W1 Cells for Rapid Evaluation of Drinking Water Samples for Toxicity

This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity ...

Administering and Detecting Protein Marks on Arthropods for Dispersal Research

Monitoring arthropod movement is often required to better understand associated population dynamics, dispersal patterns, host plant preferences, and other ...

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The need to replace petroleum fuels with alternatives from renewable and more environmentally sustainable sources is of growing importance. ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Experimental System of Solar Adsorption Refrigeration with Concentrated Collector

To improve the performance of solar adsorption refrigeration, an experimental system with a solar concentration collector was set up and investigated. The ...

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...