eoe sub articles

EoE Sub Articles

1. Preparation of the Immunogens
<ol>
	<li>Grow each cyanobacterial strain in the corresponding culture medium under conditions described in&#160;Table 1.&#160;&#160; 
	NOTE: Growth medium and culture conditions for each&#160;cyanobacteria&#160;strain are listed in&#160;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&#160;Planktothrix rubescens&#160;was generated from a natural sample of the monospecific bloom of this cyanobacterium from the Vilasouto reservoir (northern Spain).</li>
	<li>Quantify the number of cells using a cell counting chamber by optical microscopy to obtain approximately 108&#160;cells/mL from a late exponential or stationary growth phase culture.</li>
	<li>Harvest cells from 5 mL of culture by centrifugation at 2,000 x g for 5 min.</li>
	<li>Discard the supernatant and resuspend the cells in a 10-mL tube with 5 mL of 1x phosphate-buffered saline (1x PBS) to obtain around 108&#160;cells/mL.</li>
	<li>Homogenize and lyse the cells of the suspension by sonication for 5 cycles, 30 s each, with a 30- to 60-s pause on ice, using a portable, hand-held ultrasonic processor or by dipping the tube into the water bath of a cell disruptor at maximum amplitude (30 kHz).</li>
	<li>Repeat steps 1.3 - 1.5 for each strain of cyanobacterium.&#160;&#160;&#160;&#160;&#160; 
	NOTE: Sonication produces cell disruption and cellular content release. While proteins and polysaccharides are good immunogens, specific molecules, such as lipids and nucleic acids, do not induce a humoral immunogenic response by themselves. Therefore, they must bind to carriers, such as polysaccharides or proteins, to increase the molecular complexity. In addition, sonication releases intracellular material that can trigger antibody production.</li>
</ol>
2. Production of Polyclonal Antibodies
<ol>
	<li>Prepare the first immunogen dose by mixing 0.5 mL of the ultrasonicated cell lysate obtained in step 1.5 with 0.5 mL of complete Freund&#39;s adjuvant. Deliver it to the operator of an animal facility for polyclonal rabbit antibody production.</li>
	<li>Prepare three more doses as before for further use as memory boosts in the antibody production process by mixing 0.5 mL of the same homogenate/lysate obtained in step 1.5 with 0.5 mL of incomplete Freund&#39;s adjuvant. Give them to the animal facility to fulfill the antibody production process.</li>
	<li>Repeat steps 2.1 and 2.2 for each new antibody production process.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Normally, antibody production is entrusted to specialized animal facilities or companies because appropriate licensing and training are required to work with animals. The companies supply a relative enzyme-linked immunosorbent assay (ELISA) measurement of the quantity of antigen-specific antibodies present in the serum sample.</li>
</ol>
3. Antibody Purification
<ol>
	<li>Purify the immunoglobulin G (IgG) fraction from both the immune and the pre-immune serum collected in step 2 by protein-A affinity chromatography. Some commercial purification kits, based on cartridge systems, work well; follow the provider instructions.</li>
	<li>When the purification kit does not provide a desalting system, change the buffer after the elution of the purified antibodies to 0.1x PBS, either by dialysis or by using centrifugal filter devices with a 100-kDa (or lower) membrane pore size.</li>
	<li>Determine the antibody concentration by measuring the absorbance at 280 nm or by using colorimetric methods, such as Bradford, Lowry, or Bicinchoninic acid (BCA). 
	NOTE: It is important to avoid including amine groups in the elution buffer (e.g.,&#160;Tris buffers) because they compete with the antibody for binding to solid surfaces activated with epoxy groups. After purification, it is important to test the antibody activity with ELISA.</li>
</ol>
4. Fluorescence Antibody Labeling
<ol>
	<li>Label the purified antibodies obtained in section 3 with a fluorochrome (e.g.,&#160;a far-red fluorescent dye) by dissolving a commercial vial containing dye for labeling 1 mg of protein into 100 &#181;L of dimethyl sulfoxide (DMSO). Add 2 &#181;L of the dissolved dye to each antibody preparation at a concentration of 2 mg/mL in a final volume of 50 &#181;L in 50 mM phosphate-buffered saline (pH 8.5).</li>
	<li>Maintain the labeling reactions under continuous agitation for 1 h at ambient temperature and 1,200 rpm on a vibrating platform.</li>
	<li>Purify the labeled antibodies by size exclusion chromatography (e.g.,&#160;using a gel with a fractionation range between 1.5 and 30 kDa trapped into a column), following supplier recommendations.</li>
	<li>Measure the absorbance at 280 nm and at 650 nm in eluates and calculate the labeling efficiencies following supplier recommendations.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Up to 50 x 50-&#181;L antibody-labeling reactions can be done with a single vial of the fluorescent dye for labeling 1 mg of protein. It is recommended to cover the tubes with aluminum foil or, alternatively, to use opaque 0.5-mL tubes to avoid quenching processes after step 4.3. For IgG antibodies, optimal labeling is achieved with 3-7 mol of the dye per mol of antibody.</li>
</ol>
5. CYANOCHIP Production
<ol>
	<li>Antibodies and controls in printing solution
	<ol>
		<li>Prepare 30 &#181;L of each purified antibody in printing solution by mixing each antibody at 1 mg/mL in a commercial 1x protein printing buffer with 0.01% (v/v) Tween 20 (a non-ionic detergent), all as final concentrations. 
		NOTE: Alternatively, an antibody solution may contain 20% glycerol, 1% (w/v) sucrose (or trehalose, as a preservative), and 0.01% (v/v) Tween 20 in carbonate buffer (pH 8.5).</li>
		<li>Prepare 30 &#181;L of the printing solution as controls: (a) 1x protein printing buffer with 0.01% (v/v) Tween 20, (b) protein-A purified pre-immune serum at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20, and (c) bovine serum albumin (BSA) at 1 mg/mL in 1x protein printing buffer with 0.01% (v/v) Tween 20.</li>
		<li>Prepare 30 &#181;L of a fluorescently labeled, purified pre-immune serum at different concentrations (e.g.,&#160;from 50 &#181;g/mL to 1 &#181;g/mL) in 1x protein printing buffer with Tween 20, as in step 5.1.1. These samples will be used as fluorescent frame markers and for relative fluorescence quantification after spotting.</li>
		<li>Add 30 &#181;L per well of the printing solutions prepared in steps 5.1.1, 5.1.2, and 5.1.3 to the highest-quality 384-well microplates for microarray manufacturing, such as polypropylene.&#160; 
		NOTE: Protein printing buffer increases the quality and stability of the antibodies, and Tween 20 homogenizes spot morphology and builds up protein coupling up. It is recommended to maintain the 384-well microplate at 4 &#176;C before use. Store it at -20 &#176;C for long periods of time. Polypropylene has low DNA, protein, cell extract, and small-molecule intrinsic binding.</li>
	</ol>
	</li>
	<li>Antibody printing onto a microscope slide 
	NOTE: Print the antibodies onto activated microscope slides by using different array platforms, like contact, split-needle, or non-contact devices, such as piezoelectric printers or &#34;ink jet&#34; technologies. In this work, the CYANOCHIP has been routinely printed by contact using a robotic system (arrayer) capable of spotting nL quantities of the antibodies at the &#181;m scale.
	<ol>
		<li>Set the environmental conditions of the printing room to 20 &#176;C and 40 - 50% relative humidity.</li>
		<li>Set up the slide substrates (e.g.,&#160;75- x 25-mm epoxy-activated microscope glass slides) to perform several identical antibody arrays on each slide.</li>
		<li>Spot each purified antibody, including controls and the reference frame, in a triplicate spot pattern; under these conditions, the spots are 180 - 200 &#181;m in diameter.</li>
		<li>After printing, leave the slides for at least 30 min at ambient temperature to let them dry, and then store them at 4 &#176;C; for working in the field, the slides can be transported and stored at ambient temperature for several months. 
		NOTE: The CYANOCHIP is printed in a triplicate spot microarray format with 3 x 8 identical microarrays per slide or 9 identical arrays in a 1 x 9 microarray format. Each array size must not be higher than the reaction chamber dimensions for a 24-well gasket (usually 7.5 x 6.5 mm in a 3 x 8 hybridization chamber).</li>
		<li>Before using the microarray to analyze environmental samples, use fluorescent sandwich microarray immunoassay (FSMI) to determine the working dilution for each antibody in a titration curve. For each antibody, use a standard concentration of the corresponding immunogen (103&#160;- 104&#160;cells/mL) and serial dilutions of the fluorescent antibody (between 1:500 and 1:32,000). The optimal antibody concentration corresponds to 50% of the maximum signal intensity obtained in the titration curve. Also, the sensitivity and specificity for each antibody must be determined, as described in Blanco&#160;et al.</li>
	</ol>
	</li>
</ol>
6. Preparation of Environmental Multianalyte Extracts for the Fluorescent Sandwich Microarray Immunoassay (FSMI)
<ol>
	<li>Multianalyte extract from a liquid sample
	<ol>
		<li>Take 1 - 100 mL of the liquid sample with a sterile syringe (e.g.,&#160;water from the shore of a water reservoir); the amount of sample is greatly dependent upon the potential concentration of the targets.</li>
		<li>Concentrate the cells by passing the water sample through a 3-&#181;m pore size, 47-mm diameter polycarbonate filter; a cellular concentration between 103&#160;- 108&#160;cells/mL is desirable for positive detection, but the actual concentration is unknown.</li>
		<li>Recover the biomass collected in the filter with 1 mL of a modified Tris-buffered saline, Tween 20-reinforced buffer (TBSTRR; 0.4 M Tris-HCl (pH 8), 0.3 M NaCl, and 0.1% Tween 20) by scraping it with a spatula into a 15-mL tube.</li>
		<li>Homogenize and disaggregate by using a hand-held ultrasonic processor, as described in step 1.5, or just by pipetting up and down multiple times; this prepares the sample for analysis by the microarray.</li>
	</ol>
	</li>
	<li>Multianalyte extract from a solid sample
	<ol>
		<li>Weigh up to 0.5 g of the solid sample (e.g.,&#160;rock, soil, or sediments) into a 10-mL tube and add up to 2 mL of TBSTRR.</li>
		<li>Sonicate by immersing the sonicator probe in the tube, by dipping the tube into the water bath of a powerful sonicator horn, or by using a hand-held sonicator. Perform at least 5 x 30-s cycles at 30 kHz, stopping for 30 s while on ice.</li>
		<li>Filter to remove sand, clay, and other coarse material with a 10-mL syringe coupled to a 10- to 12-mm diameter, 5- to 20-&#181;m pore size nylon filter holder. Push the sample through the filter into a 1.5-mL tube. If the filter saturates, agitate the suspension in the syringe and take it to a new one; this prepares the filtrate material for the immunoassay (step 7).&#160;&#160;&#160;&#160;&#160; 
		NOTE: The buffering capacity of TBSTRR depends on the type of sample. It is important to carry out the immunoassay immediately after the preparation of the environmental extract to avoid the effect of enzymatic degradation on the analytes. Alternatively, add protease inhibitors into the environmental extract and freeze at -80 &#176;C until the next step.</li>
	</ol>
	</li>
</ol>
7. Fluorescent Sandwich Microarray Immunoassay (FSMI)
<ol>
	<li>Blocking the CYANOCHIP&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Immediately before use, treat the printed slides to block all free epoxy groups on the slide and to remove the excess of non-covalently bound antibodies.
	<ol>
		<li>Immerse the microarray into 0.5 M Tris-HCl (pH 9) with 5% (w/v) BSA solution on a clean surface (e.g.,&#160;Petri dish or a 50-mL tube) with mild agitation from a rocker platform for 5 min. Alternatively, lay the slide down onto a 100- to 200-&#181;L drop of the above solution with the microarray spots facing it. Leave for 3 - 5 min and then proceed.</li>
		<li>Carefully pick up the slide using plastic-tipped forceps; try to avoid touching microarray zones. Eliminate the excess of liquid by softly knocking the slide onto a paper towel. Immerse it in 0.5 M Tris-HCl (pH 8) with 2% (w/v) BSA solution for 30 min with mild agitation from a rocker platform.</li>
		<li>Dry the slide by performing a short centrifugation (200 - 300 x g for 1 min) using a commercial microcentrifuge adapted for microscope slides. Alternatively, dry the slide by softly knocking onto a paper towel.</li>
	</ol>
	</li>
	<li>Incubation of the multianalyte sample extract with the microarray
	<ol>
		<li>Set up the slide in a commercial microarray hybridization cassette with 24 wells for multiple microarrays; follow the provider instructions.</li>
		<li>After slide and cassette assembly, pipette up to 50 &#181;L of the sample extract or a dilution of it in TBSTRR into each well of the cassette.</li>
		<li>Repeat step 7.2.2 for each sample to be analyzed.</li>
		<li>As a blank control, pipette 50 &#181;L of TBSTRR buffer into at least two separate wells of the cassette.</li>
		<li>Incubate at ambient temperature for 1 h with mixing by pipetting every 15 min or by leaving it under mild shaking. Alternatively, incubate for 12 h at 4 &#176;C. 
		NOTE: Use other incubation gaskets as a function of the microarray pattern. The time and temperature of the incubation in step 7.2.5 are empirical parameters that normally depend on the affinity and binding kinetics of each paired antigen-antibody.</li>
	</ol>
	</li>
	<li>Washing
	<ol>
		<li>Remove the samples by putting the cassette down and carefully knocking it onto a clean, absorbent paper.</li>
		<li>Wash the wells by adding 150 &#181;L of TBSTRR to each one, and eliminate the buffer, as above.</li>
		<li>Repeat step 7.3.2 three more times.</li>
	</ol>
	</li>
	<li>Incubation with fluorescent detector antibodies
	<ol>
		<li>Add 50 &#181;L of an antibody mixture containing the 17 anti-cyanobacterial-strain antibodies, each labeled with the fluorochrome in TBSTRR with 1% (w/v) BSA. Determine the concentration of each fluorescent antibody in the mixture (from 0.7 to 2 &#181;g/mL)&#160;by performing titration experiments of each antigen/antibody pair.</li>
		<li>Incubate for 1 h at ambient temperature, as described in step 7.2.5, or for 12 h at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Washing out the fluorescent antibodies
	<ol>
		<li>Remove the fluorescent unbound antibodies, as in step 7.3.</li>
		<li>Disassemble the cassette and immerse the slide into 0.1x PBS (e.g.,&#160;in a 50-mL tube) for a quick rinse.</li>
		<li>Dry the slide as in step 7.1.3.</li>
	</ol>
	</li>
</ol>
8. Scanning for Fluorescence
<ol>
	<li>Scan the slide for fluorescence at the maximum emission fluorescence peak for far-red fluorescent dye in a scanner for fluorescence. Take several images of the microarrays at different scanning parameters, generally by lowering the laser gain value. 
	NOTE: Avoid saturated spots (&#62; 65,000 fluorescence counts)&#8212;they are out of scale and may introduce quantification errors.</li>
</ol>
Table 1.&#160;List of the Antibodies (Abs) and the Cyanobacterial Strains Used to Produce the CYANOCHIP.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ab code</td>
			<td>Immunogen (strain)</td>
			<td>Order</td>
			<td>Habitat</td>
			<td>Medium</td>
			<td>Culture conditions</td>
		</tr>
		<tr>
			<td>K1</td>
			<td>Anabaena sp.</td>
			<td>Nostocales</td>
			<td>unknown</td>
			<td>BG11 and nitrate</td>
			<td>30 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K2</td>
			<td>Anabaena sp.</td>
			<td>Nostocales</td>
			<td>unknown</td>
			<td>BG11o</td>
			<td>30 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K3</td>
			<td>Microcystis flosaquae</td>
			<td>Chroococcales</td>
			<td>planktonic</td>
			<td>BG11</td>
			<td>28 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K4</td>
			<td>Microcystis novacekii</td>
			<td>Chroococcales</td>
			<td>planktonic</td>
			<td>BG11</td>
			<td>28 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K5</td>
			<td>Microcystis aeruginosa</td>
			<td>Chroococcales</td>
			<td>planktonic</td>
			<td>BG11</td>
			<td>28 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K6</td>
			<td>Aphanizomenon ovalisporum</td>
			<td>Nostocales</td>
			<td>planktonic</td>
			<td>BG11o</td>
			<td>28 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K7</td>
			<td>Phormidium sp.</td>
			<td>Oscillatoriales</td>
			<td>benthic</td>
			<td>BG11</td>
			<td>18 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K8</td>
			<td>Rivularia sp.</td>
			<td>Nostocales</td>
			<td>benthic</td>
			<td>CHU-D</td>
			<td>18 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K9</td>
			<td>Chamaesiphon sp.</td>
			<td>Chroococcales</td>
			<td>benthic</td>
			<td>BG11</td>
			<td>18 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K10</td>
			<td>Leptolyngbya boryana</td>
			<td>Oscillatoriales</td>
			<td>benthic</td>
			<td>BG11</td>
			<td>18 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K11</td>
			<td>Tolypothrix distorta</td>
			<td>Nostocales</td>
			<td>benthic</td>
			<td>BG11o</td>
			<td>18 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K12</td>
			<td>Aphanizomenon aphanizomenoides</td>
			<td>Nostocales</td>
			<td>planktonic</td>
			<td>BG11o</td>
			<td>28 &#186;C, continuous light</td>
		</tr>
		<tr>
			<td>K13</td>
			<td>Nostoc sp. (Antarctica)</td>
			<td>Nostocales</td>
			<td>benthic</td>
			<td>BG11o</td>
			<td>13 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K14</td>
			<td>Anabaena sp.</td>
			<td>Nostocales</td>
			<td>benthic</td>
			<td>BG11o</td>
			<td>13 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K15</td>
			<td>Leptolyngbya sp.</td>
			<td>Oscillatoriales</td>
			<td>benthic</td>
			<td>BG11</td>
			<td>13 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K16</td>
			<td>Tolypothrix sp.</td>
			<td>Nostocales</td>
			<td>benthic</td>
			<td>BG11</td>
			<td>13 &#186;C, 16-8 photoperiod</td>
		</tr>
		<tr>
			<td>K17</td>
			<td>Planktothrix rubescens</td>
			<td>Oscillatoriales</td>
			<td>planktonic</td>
			<td>none</td>
			<td>none</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 mm pore diameter filters</td>
			<td>Millipore</td>
			<td>GSWP04700</td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr>
			<td>Eppendorf&#160; 5424R microcentrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS) pH 7.4 (10X)</td>
			<td>Thermofisher Scientific</td>
			<td>70011036</td>
			<td>50 mM potassium phosphate, 150 mM NaCl, pH 7.4</td>
		</tr>
		<tr>
			<td>Ultrasonic processor UP50H</td>
			<td>Hielscher</td>
			<td></td>
			<td>For preparation of immunogens</td>
		</tr>
		<tr>
			<td>Complete Freund&#39;s adjuvant&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F5881</td>
			<td>Immunopotentiator</td>
		</tr>
		<tr>
			<td>Incomplete Freud&#39;s adjuvant</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F5506</td>
			<td>For boost injections</td>
		</tr>
		<tr>
			<td>Protein A antibody purification kit&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>PURE1A&#160;</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr>
			<td>Centrifugal filter devices MWCO&#60;100 KDa</td>
			<td>Millipore</td>
			<td>UFC510096-96K</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr>
			<td>Dialysis tubings, benzoylated</td>
			<td>Sigma-Aldrich</td>
			<td>D7884-10FT</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr>
			<td>Illustra Microspin G-50 columns&#160;</td>
			<td>GE-HealthCare</td>
			<td>GE27-5330-02</td>
			<td>For isolation of IgG</td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Sigma-Aldrich</td>
			<td>B6916-500 mL</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr>
			<td>MicroBCA protein assay kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23235</td>
			<td>To quantify the antibody concentration</td>
		</tr>
		<tr>
			<td>Protein arraying buffer 2X</td>
			<td>Whatman (Sigma Aldrich)&#160;</td>
			<td>S00537</td>
			<td>Printing buffer; 30-40% glycerol in 1X PBS with 0.01% Tween 20</td>
		</tr>
		<tr>
			<td>Tween 20&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9416</td>
			<td>Non-ionic detergent</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9418</td>
			<td>Control&#160; for printing; blocking reagent</td>
		</tr>
		<tr>
			<td>384-wells microplate</td>
			<td>Genetix</td>
			<td>X6004</td>
			<td>For antibody printing</td>
		</tr>
		<tr>
			<td>Robot arrayer for multiple slides</td>
			<td>MicroGrid II TAS arrayer from Digilab</td>
			<td></td>
			<td>For antibody printing</td>
		</tr>
		<tr>
			<td>Epoxy substrate glass slides</td>
			<td>Arrayit corporation</td>
			<td>VEPO25C</td>
			<td>Solid support for antibody printing</td>
		</tr>
		<tr>
			<td>Alexa Fluor-647 Succinimidyl-ester&#160;</td>
			<td>Molecular probes</td>
			<td>A20006</td>
			<td>Fluorochrome</td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D8418</td>
			<td>Fluorochrome dissolvent</td>
		</tr>
		<tr>
			<td>Heidolph Titramax vibrating platform shaker</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>For antibody labeling&#160;</td>
		</tr>
		<tr>
			<td>Illustra Microspin G-50 columns&#160;</td>
			<td>Healthcare</td>
			<td>27-5330-01</td>
			<td>For purification of labeled antibodies</td>
		</tr>
		<tr>
			<td>Safe seal brown 0,5 ml tubes</td>
			<td>Sarstedt</td>
			<td>7,27,04,001</td>
			<td>For labeled antibodies storage&#160;</td>
		</tr>
		<tr>
			<td>Nanodrop 1000 spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>To quantify antibody concentration and labeling efficiency</td>
		</tr>
		<tr>
			<td>3 &#181;m pore size polycarbonate 47 mm diameter filter</td>
			<td>Millipore</td>
			<td>TMTP04700</td>
			<td>To concentrate cells</td>
		</tr>
		<tr>
			<td>1M Trizma hydrochloride solution pH 8</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T3038</td>
			<td>For TBSTRR preparation; to block slides</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S7653</td>
			<td>For TBSTRR preparation</td>
		</tr>
		<tr>
			<td>20 &#181;m nylon filters&#160;</td>
			<td>Millipore</td>
			<td>NY2004700</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr>
			<td>10-12 mm filter holders</td>
			<td>Millipore</td>
			<td>SX0001300</td>
			<td>For environmental extract preparation</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P8340</td>
			<td>For environmental extract storage</td>
		</tr>
		<tr>
			<td>1M Trizma hydrochloride solution pH 9</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T2819</td>
			<td>To block slides</td>
		</tr>
		<tr>
			<td>Heidolph Duomax 1030 rocking platform shaker</td>
			<td>VWR</td>
			<td></td>
			<td>To block slides; for incubation processes&#160;</td>
		</tr>
		<tr>
			<td>VWR Galaxy miniarray microcentrifuge</td>
			<td>VWR</td>
			<td>C1403-VWR</td>
			<td>To dry slides</td>
		</tr>
		<tr>
			<td>Multi-Well microarray hybridization cassette</td>
			<td>Arrayit corporation</td>
			<td>AHC1X24</td>
			<td>Cassette for 24 assays per slide</td>
		</tr>
		<tr>
			<td>GenePix 4100A microarray scanner&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Scanner for fluorescence</td>
		</tr>
		<tr>
			<td>GenePix Pro Software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>Software for image analysis and quantification</td>
		</tr>
	</tbody>
</table>

a technique to detect cyanobacterial strains using an antibody microarray chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

1. Preparation of the Immunogens

	Grow each cyanobacterial strain in the corresponding culture medium under conditions described in&#160;Table ...

Begin with an antibody microarray chip printed with specific antibodies.
Each spot contains a unique antibody against a specific cyanobacterial strain.
Treat the chip with a blocking buffer to minimize non-specific interactions.
Assemble the chip in a hybridization cassette possessing multiple wells.
Introduce an environmental sample into the wells and incubate it under optimized conditions.
The sample contains various bacteria, including different cyanobacterial strains, that interact with specific antibodies on the printed chip. Wash the chip to remove the unbound bacteria.
Add an antibody mixture containing fluorophore-labeled anti-cyanobacterial-strain antibodies interacting with specific antigens on the respective cyanobacterial cells. Remove unbound antibodies.
Disassemble the cassette, wash and dry the chip, then scan it.
Fluorescence in specific spots signifies the presence of particular cyanobacterial strains in the sample.

a-technique-to-detect-cyanobacterial-strains-using-an-antibody

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

58 Views. Source: Blanco, Y. et al., Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip. J. Vis. Exp. (2017) This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strai...

Video: A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip - Concept

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

JoVE Journal

Chemistry

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of cyanobacteria and related cyanotoxins in water samples and in an organic matrix, such as bivalve extracts. FDS combines remote/proximal sensing techniques with analytical/bioinformatics analyses. Sampling spots are chosen through multi-disciplinary, multi-scale, and multi-parametric monitoring in a three-dimensional physical space, including remote sensing. Microscopic observation and taxonomic analysis of the samples are performed in the laboratory setting, which allows for the identification of cyanobacterial species. Samples are then extracted with organic solvents and processed with LC-MS/MS. Data obtained by MS/MS are analyzed using a bioinformatic approach using the online platform Global Natural Products Social (GNPS) to create a network of molecules. These networks are analyzed to detect and identify toxins, comparing data of the fragmentation spectra obtained by mass spectrometry with the GNPS library. This allows for the detection of known toxins and unknown analogues that appear related in the same molecular network.

A fast-multidisciplinarystrategy for early detection of cyanobacterial blooms and associated cyanotoxins is described here. It allows for the detection of cyanobacteria and related cyanotoxins in water samples and in organic matrices, such as bivalve samples, in 24 h.

Fast detection of cyanobacteria and cyanotoxins is achieved using a Fast Detection Strategy (FDS). Only 24 h are needed to unravel the presence of ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Transcript

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Early Detection of Cyanobacterial Blooms and Associated Cyanotoxins using Fast Detection Strategy

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays