We appreciate the colleagues in Taiwan Cetacean Society, Marine Biology and Cetacean Research Center of National Cheng Kung Univerisy, Farglory Ocean Park, and Animal Disease Diagnostic Center of National Chiayi Universiy for sample collection. This project was funded by grant to WCY from the Council of Agriculture of Taiwan (100AS-02.1-FB99).

Ethics Statement: The study was performed in accordance with international guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of National Chiayi University, approval ID: 99022. The cetacean sample use was permitted by Council of Agriculture of Taiwan (Research Permit 100M-02.1-C-99).
1. Muscle Sample Preparation and SDS-PAGE
Note: Muscle samples from 23 species including 16 species of marine mammals, 5 species of terrestrial mammals, tuna and chicken were used in this study (Table 1). The cetacean muscle samples were obtained from stranded individuals, fishery bycatch, and confiscation. Rabbit, rat, dog, and chicken muscle tissues were obtained from Animal Disease Diagnostic Center of National Chiayi University. Samples of beef, pork, lamb, and tuna were purchased from a local supermarket. The muscle sample of harbor seal (Phoca vitulina) was provided by Farglory Ocean Park. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate soluble proteins with different molecular weights in muscle samples.
<ol>
	<li>Store all samples at -20 &#176;C until use.</li>
	<li>Pre-cool mortar at -20 &#176;C. Then put 3 g of frozen muscle sample into it.</li>
	<li>Homogenize the sample with 10 ml of cool phosphate-buffered saline (PBS) using a tissue homogenizer.</li>
	<li>Centrifuge the homogenized sample at 10,000 x g for 10 min at 4 &#176;C. Collect the supernatants and store it at -20 &#176;C until use.</li>
	<li>Prepare 5 ml of 5% stacking gel (3.07 ml of distilled water, 1.25 ml of 4x upper gel buffer of pH 6.8, 625 &#181;l of 40% acrylamide, 50 &#181;l of 10% ammonium persulfate (APS), and 5 &#181;l of tetramethylethylenediamine (TEMED)) and 10 ml of 15 % separating gel (3.65 ml of distilled water, 2.5 ml of 4x lower gel buffer of pH 8.8, 3.75 ml of 40% acrylamide, 100 &#181;l of 10% APS, and 4 &#181;l of TEMED).
	<ol>
		<li>In the electrophoresis cell, pour stacking gel (5% acrylamide) on top of the separating gel (15% acrylamide) after the latter has solidified. Insert a gel comb in the stacking gel.</li>
	</ol>
	</li>
	<li>Perform PAGE according to the following conditions: initial run condition: 100 volts, 20 min and final condition: 120 volts, 40 min.</li>
	<li>Stain the gel with Coomassie brilliant blue at room temperature for 30 min until the gel is uniformly blue in color. 
	Note: Staining is complete when the gel is no longer visible in the dye solution.</li>
	<li>Destain it with acetic acid solution (10%) at room temperature for 1 hr. Bands will begin to appear. Continue destaining at 4 &#176;C overnight until background is clear.</li>
</ol>
2. Peptide Synthesis and Monoclonal Antibody Production
<ol>
	<li>Retrieve the amino acid sequences of Mb from GenBank including tuna, chicken, ostrich, domestic mammals, seal and 18 species of cetaceans (Table 2).</li>
	<li>Align the sequences using proper software16:
	<ol>
		<li>Launch the Alignment Explorer by selecting the Align: Edit/Build Alignment on the launch bar; select Create New Alignment and click OK. A dialog will appear asking &#34;Are you building a DNA or Protein sequence alignment?&#34;</li>
		<li>Click the button labeled &#34;Protein&#34;; select Data: Open: Retrieve sequences from File and select sequence file; select the Edit: Select All menu command to select all sites for every sequence in the data set for creating a multiple sequence alignment.</li>
		<li>Select Alignment: Align by ClustalW from the main menu to align the selected sequences data using the ClustalW algorithm; select &#34;BLOSUM&#34; as the Protein Weight Matrix then click the OK button.</li>
	</ol>
	</li>
	<li>Analyze the sequence alignment:
	<ol>
		<li>Focus on 5 antigenic reactive sites17: site 1 (AKVEADVA, 15-22), site 2 (KASEDLK, 56-62), site 3 (ATKHKI, 94-99), site 4 (HVLHSRH, 113-119) and site 5 (KYKELGY, 145-151) and find the fragment conserved among cetaceans. An * (asterisk; consensus symbol in the alignment (Protocol 2.2.3)) indicates positions which have a single, fully conserved residue. The following conserved fragments in cetaceans were found: sequence KASEDLKKHG (which includes site 2) and sequence HVLHSRHP (which includes site 4).</li>
	</ol>
	</li>
	<li>Synthesize candidate sequence fragments according to the sequence analysis, and conjugate with an ovalbumin protein (OVA) as carrier protein using commercial services.
	<ol>
		<li>Add hydrophobic amino acids (e.g., methionine) to the N-terminal of antigenic reactive site for preventing the peptide from decomposition (e.g., M-KASEDLKKHG).</li>
		<li>Lengthen the C-terminal of antigenic reactive site for exposing the core antigenic site to the immunocyte. Furthermore, conjugate the peptide with OVA by adding a cysteine (Cys, C) to the C-terminal (e.g., M-KASEDLKKHG-NTVL-C).</li>
	</ol>
	</li>
	<li>Emulsify complete Freund&#39;s adjuvant for immunogen 1 or incomplete Freund&#39;s adjuvant for immunogen 2 with an equal volume of each synthetic peptide in PBS (3 ml, final concentration 30-50 &#181;g/100 &#181;l).</li>
	<li>Inoculate immunogen 1 (0.1 mg) subcutaneously into each of 5 female BALB/c mice.</li>
	<li>Perform subcutaneous booster injections five times using immunogen 2 at two-week intervals and collect test sera by tail clip sampling from the mice before every booster.</li>
	<li>Determine sera titer for the first screening.
	<ol>
		<li>Dissolve 100 &#181;g of free peptide in 25 ml of reaction buffer and aliquot 50 &#181;l of the solution into each well of a 96-well plate. Add 10 &#181;l coupling reagent solution into each well and mix the plate. Incubate the plate for 2 hr at room temperature.</li>
		<li>Remove the contents of the well, wash each well 3 times with distilled water, and block the plate by adding 200 &#181;l of blocking solution. Incubate for 1 hr at room temperature. Remove the contents and wash each well 3 times with distilled water.</li>
		<li>Perform indirect ELISA (Protocol 5.1-5.7) for determining sera titer for the first screening. Choose the mouse with the highest titer (highest optical density) for spleen collection.</li>
	</ol>
	</li>
	<li>Three days prior to spleen collection, inoculate 0.1 mg of immunogen 1 subcutaneously into the mouse presenting the highest titer.</li>
	<li>Collect the spleen cells from the selected immunized mice and fuse with murine myeloma cells F0 (sp2/0-Ag14) to obtain hybridoma cells for mAb generation18.</li>
	<li>14 days after fusion, select the positive clones by screening the reactivity of hybridoma supernatants toward free synthetic peptide using indirect ELISA (Protocol 5.1-5.7).</li>
	<li>Dilute the cells to an appropriate number per well for maximizing the proportion of wells that contain only one single clone (dilution cloning).
	<ol>
		<li>Add 100 &#181;l of cell culture medium to all the wells in the 96-well plate except well A1 which is left empty.</li>
		<li>Add 200 &#181;l of the cell suspension to well A1. Then quickly transfer 100 &#181;l from A1 to B1 and mix by gently pipetting. Repeat these 1:2 dilutions down the entire column, and then discard 100 &#181;l from H1 so that it ends up with the same volume as the wells above it.</li>
		<li>Add an additional 100 &#181;l of medium to column 1 with an 8-channel micropipette. Then quickly transfer 100 &#181;l from each of the wells in column 1 to those in column 2 using the same pipette, and mix by gently pipetting.</li>
		<li>Using the same tips, repeat these 1:2 dilutions across the entire plate. Discard 100 &#181;l from each of the wells in the last column.</li>
		<li>Bring the final volume of all wells to 200 &#181;l by adding 100 &#181;l medium to each well. Incubate plate undisturbed at 37 &#176;C in a humidified CO2 incubator.</li>
		<li>Check each well and mark all wells that contain just a single colony. Carry out two or more clonings until &#62;90% of the wells containing single clones are positive for antibody production.</li>
	</ol>
	</li>
	<li>Screen the clones by western blot and dot blot (protocol 3.1-4.6). Then subculture colonies from the wells into larger vessels to expand cells for obtaining mAb. Usually each clone is transferred into a single well in a 12- or 24-well plate.</li>
	<li>Measure the affinity between the mAbs and the muscle extracts from cow, goat, pig, dog, rabbit, tuna, chicken, seal, and four representative cetacean species by western blot and dot blot (protocol 3.1-4.6).</li>
	<li>Inoculate selected hybridoma cells (up to 3 x 106) intraperitoneally into mice to induce ascites. Abdominal swelling is typically apparent within 7-10 days post hybridoma injection. Collect fluid using a hypodermic needle (less than 20 gauge).</li>
	<li>Centrifuge ascites fluid (10,000 x g for 10 min) to remove cells and debris. Filter through a 0.45 &#181;m filter. Add 1 to 20 ml of the sample, 15 ml binding buffer, and 3 to 5 ml of elution buffer into the protein G Sepharose column. Collect the elution fraction containing purified antibody from the mice ascites fluid.</li>
	<li>Determine the antibody isotype by antibody isotyping kit using manufacturer&#39;s instructions.</li>
</ol>
3. Western Blot
<ol>
	<li>Prepare 2x loading buffer containing &#946;-mercaptoethanol (BME) by mixing 950 &#181;l of 2x Laemmli sample buffer with 50 &#181;l of BME. Dilute the muscle supernatants (protocol 1.1-1.4) in loading buffer with appropriate ratio for obtaining good signals: 1:50 (cetaceans and seal) and 1:5 (domestic animals and tuna) when using polyclonal rabbit anti-human Mb antibody, and 1:1 (pig, rabbit, chicken and tuna), 1:5 (cow, goat and dog), and 1:25 (cetaceans and seal) when antibody is from hybridoma supernatant.</li>
	<li>Heat sample at 95 &#176;C for 5 min. Load samples into the wells of SDS-PAGE gel (4% acrylamide stacking and 12% acrylamide separating) along with molecular weight markers. Run the gel for 5 min at 50 V then increase the voltage to 150 V to finish the run in about 1 hr.</li>
	<li>Place the gel in 1x transfer buffer for 15 min. Transfer the separated protein to nitrocellulose (NC) membranes after they are separated by PAGE. Transfer can be done at 100 V for 60-90 min.</li>
	<li>Prepare blocking solution: 1x PBS containing 0.1% Tween 20 with 5% nonfat dry milk. Block the NC membrane in 25 ml of blocking solution at room temperature for 1 hr. Wash three times for 5 min each with 15 ml phosphate-buffered saline with Tween 20 (PBST).</li>
	<li>Incubate the membrane and primary antibody (ascites fluid or hybridoma supernatant) in 10 ml antibody dilution buffer diluted with 5% blocking solution at 4 &#176;C overnight.</li>
	<li>Wash the membrane three times again with PBST to clean off excessive antibody.</li>
	<li>Incubate the membrane with alkaline phosphatase-conjugated goat anti-mouse IgG at 1:1,250 in blocker solution with gentle agitation for 1 hr at room temperature.</li>
	<li>Wash the membrane again and incubate it in the 5-bromo-4-chloro-3-indolyl phosphate/p-nitroblue tetrazolium chloride (BCIP/NBT) phosphatase substrate mixture for 10 to 20 min until color development.</li>
	<li>Stop the reaction by washing the membrane in several changes of distilled water.</li>
</ol>
4. Dot Blot
<ol>
	<li>Dilute the muscle supernatants (protocol 1.1-1.4) in 5% bovine serum albumin (BSA) in PBST with appropriate ratio for obtaining good signals: 1:5 for domestic animals and tuna, and 1:25 for cetaceans and seal. Spot 5 &#181;l of samples onto membrane. Minimize the area that the solution penetrates (usually 3-4 mm diameter) by applying it slowly.</li>
	<li>Dry the membrane at room temperature (e.g., in a laminar flow for 30-60 min), block it with blocking solution in Petri dish for 1 hr at room temperature, and wash it with PBST.</li>
	<li>Incubate the membrane with primary antibody (mAb from hybridoma supernatant at 1:10,000 or ascites fluid at 1:100,000 diluted in 5% blocking solution) for 1 hr at room temperature.</li>
	<li>Use PBST to wash the membrane three times for 5 min each to remove excess antibody, and then incubate it with secondary antibody (alkaline phosphatase-conjugated goat anti-mouse IgG at 1:1,250 in 5% blocking solution) for 1 hr at room temperature.</li>
	<li>Wash the membrane again and incubate it in the BCIP/NBT phosphatase substrate mixture within 10 to 20 min until color development.</li>
	<li>Stop the reaction by washing the membrane in several changes of distilled water.</li>
</ol>
5. Indirect ELISA
<ol>
	<li>Prepare washing buffer (0.002 M imidazole buffered saline with 0.02% Tween 20). Wash the plate 3 times with washing buffer between each following step (protocol 5.2-5.5).</li>
	<li>Prepare 1:25 dilution of muscle supernatants (protocol 1.1-1.4) in coating buffer. Coat a 96-well ELISA plate with 100 &#181;l diluted supernatants at 4 &#176;C overnight and block it with blocking buffer (1% BSA in PBS) for 1 hr at room temperature.</li>
	<li>Prepare 1:2,000 dilution of the purified mAb with diluted buffer and add 100 &#181;l of diluted mAb to each well. Incubate the plate for 1 hr at room temperature.</li>
	<li>Add goat anti-mouse IgG conjugated with horseradish peroxidase (1:200 dilution in diluted buffer) for further incubation.</li>
	<li>Add peroxidase substrate to each well (100 &#181;l/well) and incubate for 10-15 min.</li>
	<li>Stop the enzyme reaction by the peroxidase stop solution (100 &#181;l/well) when color development is observed.</li>
	<li>Read the optical density at 450 nm using a microplate spectrophotometer.</li>
</ol>
6. Preparation of Colloidal Gold-labeled mAb
Note: The color of colloidal gold solution and the mixture should always be red. Adjust pH, concentration of mAb, centrifuge speed when black precipitate is noticed. Steps 6.1 and 6.2 are optimization steps.
<ol>
	<li>Add purified detecting mAb (50 &#181;g/ml, 3 &#181;l) to 100 &#181;l of colloidal gold solution with pH values varying from 5-9. The minimum pH that keeps the color red for two hours is considered as the optimum pH. Note: In this study, 0.1 M potassium carbonate was used to adjust colloid gold (40 nm) solution to pH 8.0 (optimum pH).</li>
	<li>Add various amount of purified detecting mAb (500 &#181;g/ml, 1-20 &#181;l) to 100 &#181;l of colloidal gold solution at pH 8.0. Note: The optimum concentration in this study is 6 &#181;g/ml (no black precipitate).</li>
	<li>According to the above results, add 60 &#181;g of purified detecting mAb drop-wise to 10 ml of colloid gold solution. Emulsify the mixture gently at room temperature for 10 min. Add 2 ml of 5% BSA solution in PBS (pH 7.4) to the mixture and emulsify gently at room temperature for 15 min to reduce background interference.</li>
	<li>Centrifuge the mixture at 10,000 x g for 30 min at 4 &#176;C.</li>
	<li>Remove the supernatant with unconjugated antibody carefully and suspend the resulting pellets in 4 ml PBST containing 1% BSA and 0.1% Tween 20, and repeat centrifugation and suspension several times.</li>
	<li>Suspend the final precipitates in 1 ml PBST and store it at 4 &#176;C until used.</li>
</ol>
7. Construction of Immune Strip
Note: Figure 1 shows the immune strip design. Prepare and assemble the strips in a low-humidity laboratory environmental condition (&#60; 20% Relative Humidity) for prolonged storage life (&#62; 1 yr). The dimensions of pads and membrane are: conjugate pad 300 mm x 10 mm, absorbent pad 300 mm x 24 mm, sample pad 300 mm x 24 mm, NC membrane 300 mm x 25 mm, pasteboard 300 mm x 80 mm.
<ol>
	<li>Add colloid gold-labeled mAb solution from step 6.6 with a micropipette to saturate the conjugate pad and then dry it at 37 &#176;C for 1 hr before assembling.</li>
	<li>Distribute the specific antigen-capturing mAb (500 &#181;g/ml) on the test zone, and rabbit anti-mouse IgG (500 &#181;g/ml) on the control zone for detecting mAb on the NC membrane using a pipette or immunostrip printer. Maintain distance (&#62;5 mm) between the two zones to avoid interference.</li>
	<li>Paste the conjugated pad, absorbent pad and NC membrane on the pasteboard with double-sided tape. 
	Note: Overlap the pads on each side of the NC membrane by about 2 mm. Inappropriate strip construction will result in an incomplete test.</li>
	<li>Place the sample pad over the conjugate pad (2 mm) and paste it on the pasteboard.</li>
	<li>Create 6-mm-wide strips with a paper cutter. Pack the strips in the aluminum foil bag with desiccant, and store them at 4 &#176;C until used.</li>
</ol>
8. Cross-reactivity Test
<ol>
	<li>Homogenize 0.03 g of raw muscle sample with 1 ml PBS (containing 0.1% BSA) in a 1.5 ml centrifuge tube using a bamboo stick or grinding rod.</li>
	<li>Hold the strip by the end opposite to the test areas and dip the sample pad part into the specimen for 5-10 min and observe the result directly.
	<ol>
		<li>Optional: Collect 500 &#181;l supernatant and transfer it to a new centrifuge tube. 
		Note: This step is suggested if the signal is not obvious when the strip is directly soaked into the supernatant.</li>
	</ol>
	</li>
	<li>Test various muscle samples in triplicate. 
	Note: Here we tested tuna, chicken, seal, 15 species of cetaceans and 5 species of terrestrial mammals.</li>
	<li>Have five independent inspectors repeat 8.3 for five times, i.e., use 575 strips in total.</li>
</ol>

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The authors declare that they have no competing financial interests.

Using a synthetic peptide conjugated to carrier protein is remarkably more effective compared to its cognate protein. For a sandwich-based technique, because the mAb is developed using epitopes with known relative locations, the two mAbs in this study are not likely to interfere with each other&#39;s interaction with the target antigen epitope. Moreover, the reactivity between the native protein and the antibody of mice immunized with the synthetic peptide-conjugate may be stronger than the reactivity between the native ...

Historically, cetacean meat has been consumed in many parts of the world and this consumption continues today1. Due to the trophic level of cetaceans, high levels of mercury and other toxins are known to be present in their meat2. Therefore, the consumption of cetacean meat could lead to a health problem not only for high-risk groups such as pregnant women but also for the general population3. Furthermore, the contamination of cetacean meat with zoonotic or potentially zoonotic pathogens can also occur during its processing and storage4. It is difficult even for experienced agents to identify cetacean meats by their appearance alone. Therefore, a reliable scientific method of identification is required to differentiate cetacean meat from other meats. This would help to limit the consumption of cetacean meat.
Current methods of species identification include molecular techniques and immunological methods. Molecular techniques, such as polymerase chain reaction (PCR) and DNA sequencing, can be used to identify samples not only from raw meat5 and decomposed samples6 but also from processed foods such as cooked sausage and feedstuffs7,8. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are commonly applied in food production to detect the meat content of, for example, pork9, beef10 and catfish11. PCR-based DNA analysis for the identification of cetacean meat is available12, and has helped prevent the illegal international trade of cetacean meat in Japan, South Korea, the Philippines, Taiwan, Hong Kong, Russia, Norway, and the United States1. These methods are effective and reliable, but they can take hours or days to complete and involve laborious steps. The identification of cetacean meats is usually based on molecular techniques and there is currently no immunological method available. For regulatory agencies, it is highly desirable to develop a dependable and rapid technique that can be used in the field to identify cetacean meats. 
Immunochromatographic strips are used as detection tools with the advantage of producing rapid result via a simple protocol that is suitable for use in the field. The principles of the immunochromatographic strip and ELISA are very similar, and includes antibodies, antigens and labels. Many different labels such as colloidal gold, carbon and latex have been used in the development of immunochromatographic strips. At present, this method is commonly used for detecting antibiotics, toxin, bacteria and viruses13, but it is rarely used for identifying proteins in meat14,15. Here we propose a lateral-flow chromatographic enzyme immunoassay for rapid detection of cetacean myoglobin (Mb).

<table border="0" cellpadding="0" cellspacing="0" width="1007">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Phosphate buffered saline</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:148px;">J373</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">Protein G HP SpinTrap&#160;</td>
			<td>GE&#160;Healthcare</td>
			<td>28-9031-34</td>
			<td>spin column containing Protein G Sepharose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">IsoStrip Mouse Monoclonal Antibody Isotyping Kit</td>
			<td>Roche</td>
			<td>11493027001</td>
			<td>Isotyping Strips, precoated with subclass- and light-chain-specific anti-mouse-Ig antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini Trans-Blot</td>
			<td>Bio-Rad</td>
			<td>170-3935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrocellulose membrane</td>
			<td>Whatman</td>
			<td>Z613630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody blocker solution&#160;</td>
			<td>LTK BioLaboratories</td>
			<td style="width:148px;"></td>
			<td>To minimize nonspecific binding interactions of nonspecific IgG in the samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:512px;">BCIP/NBT phosphatase substrate&#160;</td>
			<td>KPL</td>
			<td>50-81-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Detector HRP Microwell Kit, Anti-Mouse</td>
			<td style="width:183px;">KPL</td>
			<td>54-62-18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nunc Immunoplate MaxiSorp ELISA plate</td>
			<td>Thermo&#160;Fisher Scientific</td>
			<td>EW-01928-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiskan&#160;EX ELISA reader&#160;</td>
			<td>Thermo&#160;Electron&#160;Corporation</td>
			<td>51118170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colloid gold (40 nm) solution&#160;</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td>40-50 nm is appropriate for immunostrip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td>Gibco</td>
			<td>15561-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid test immno-strip printer</td>
			<td>REGA biotechnology Inc.</td>
			<td>AGISMART RP-1000&#160;</td>
			<td>Only suited for small scale production of immunostrips for research and development purposes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Strip components (NC membranes, sample pads (#33 glass, S&#38;S), conjugate pads (#16S, S&#38;S) and absorbent pads (CF6, Whatman))</td>
			<td>REGA biotechnology Inc.</td>
			<td style="width:148px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freund&#8217;s adjuvant and incomplete Freund&#8217;s adjuvant&#160;</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:148px;">F5881, F5506</td>
			<td>Used to produce water-in-oil emulsions of immunogens</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:512px;">Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED)&#160;</td>
			<td style="width:183px;">Protech</td>
			<td style="width:148px;"></td>
			<td>Gel preparation for SDS-PAGE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie brilliant blue R-250</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610436</td>
			<td>Protein staining in SDS-PAGE gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laemmli sample buffer and &#946;-mercaptoethanol</td>
			<td style="width:183px;">Bio-Rad</td>
			<td style="width:148px;">1610737, 1610710</td>
			<td>Dilute protein samples before loading on SDS-PAGE gels</td>
		</tr>
	</tbody>
</table>

development of a colloidal gold-based immunochromatographic test strip for detection of cetacean myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Monoclonal antibody characteristics 
We developed two IgG1 mAbs (CGF5H9 and CSF1H13) recognizing two synthetic peptides (MKASEDLKKHGNTVLC and AIIHVLHSRHPAEFGC), respectively, of cetacean Mb, and these were used to construct a sandwich-type colloidal gold immunochromatographic test strip for the rapid detection of cetacean Mb. Figure 2 shows that CGF5H9 detects cetaceans and other mammals as a single stained band at a predicted molecular weight of ap...

Watch this Scientific Journal Video about Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin at JoVE.com

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

development-colloidal-gold-based-immunochromatographic-test-strip-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.1K Views.  National Chiayi University. This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals. 

Video: Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Western Blotting: Sample Preparation to Detection

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Tangential Flow Ultrafiltration: A  &ldquo;Green&rdquo; Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to their powerful antimicrobial properties.3-6 These nanoparticle applications are strongly influenced by the AgNP size and aggregation state. Many challenges exist in the controlled fabrication7 and size-based isolation4,8 of unfunctionalized, homogenous AgNPs that are free from chemically aggressive capping/stabilizing agents or organic solvents.7-13 Limitations emerge from the toxicity of reagents, high costs or reduced efficiency of the AgNP synthesis or isolation methods (e.g., centrifugation, size-dependent solubility, size-exclusion chromatography, etc.).10,14-18 To overcome this, we recently showed that TFU permits greater control over the size, concentration and aggregation state of Creighton AgNPs (300 ml of 15.3 &mu;g ml-1 down to 10 ml of 198.7 &mu;g ml-1) than conventional methods of isolation such as ultracentrifugation.19
TFU is a recirculation method commonly used for the weight-based isolation of proteins, viruses and cells.20,21 Briefly, the liquid sample is passed through a series of hollow fiber membranes with pore size ranging from 1,000 kD to 10 kD. Smaller suspended or dissolved constituents in the sample will pass through the porous barrier together with the solvent (filtrate), while the larger constituents are retained (retentate). TFU may be considered a "green" method as it neither damages the sample nor requires additional solvent to eliminate toxic excess reagents and byproducts. Furthermore, TFU may be applied to a large variety of nanoparticles as both hydrophobic and hydrophilic filters are available.
The two main objectives of this study were: 1) to illustrate the experimental aspects of the TFU approach through an invited video experience and 2) to demonstrate the feasibility of the TFU method for larger volumes of colloidal nanoparticles and smaller volumes of retentate. First, unfuctionalized AgNPs (4 L, 15.2 &mu;g ml-1) were synthesized using the well-established Creighton method22,23 by the reduction of AgNO3 with NaBH4. AgNP polydispersity was then minimized via a 3-step TFU using a 50-nm filter (460 cm2) to remove AgNPs and AgNP-aggregates larger than 50 nm, followed by two 100-kD (200 cm2 and 20 cm2) filters to concentrate the AgNPs. Representative samples were characterized using transmission electron microscopy, UV-Vis absorption spectrophotometry, Raman spectroscopy, and inductively coupled plasma optical emission spectroscopy. The final retentate consisted of highly concentrated (4 ml, 8,539.9 &mu;g ml-1) yet lowly aggregated and homogeneous AgNPs of 1-20 nm in diameter. This corresponds to a silver concentration yield of about 62%.

Tangential flow ultrafiltration (TFU) is a recirculation method used for the weight-based separation of biosamples. TFU was adapted to size-select (1-20 nm diameter) and highly concentrate a large volume of polydisperse silver nanoparticles (4 L of 15.2 &mu;g ml-1 down to 4 ml of 8,539.9 &mu;g ml-1) with minimal aggregation.

Nowadays, AgNPs are extensively used in the manufacture of consumer products,1 water disinfectants,2 therapeutics,1, 3 and biomedical devices4 due to ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

Summary

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Chapters in this video

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

Detection of Protein Ubiquitination

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

Development of automated imaging and analysis for zebrafish chemical screens.

Western Blotting: Sample Preparation to Detection

A System for ex vivo Culturing of Embryonic Pancreas

Tangential Flow Ultrafiltration: A “Green” Method for the Size Selection and Concentration of Colloidal Silver Nanoparticles

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR