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><tbody><tr><td>Phosphate buffered saline</td><td>AMRESCO</td><td>J373</td><td></td></tr><tr><td>Protein G HP SpinTrap&amp;nbsp;</td><td>GE&amp;nbsp;Healthcare</td><td>28-9031-34</td><td>spin column containing Protein G Sepharose</td></tr><tr><td>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><td>Mini Trans-Blot</td><td>Bio-Rad</td><td>170-3935</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Whatman</td><td>Z613630</td><td></td></tr><tr><td>Antibody blocker solution&amp;nbsp;</td><td>LTK BioLaboratories</td><td></td><td>To minimize nonspecific binding interactions of nonspecific IgG in the samples</td></tr><tr><td>BCIP/NBT phosphatase substrate&amp;nbsp;</td><td>KPL</td><td>50-81-00</td><td></td></tr><tr><td>Protein Detector HRP Microwell Kit, Anti-Mouse</td><td>KPL</td><td>54-62-18</td><td></td></tr><tr><td>Nunc Immunoplate MaxiSorp ELISA plate</td><td>Thermo&amp;nbsp;Fisher Scientific</td><td>EW-01928-08</td><td></td></tr><tr><td>Multiskan&amp;nbsp;EX ELISA reader&amp;nbsp;</td><td>Thermo&amp;nbsp;Electron&amp;nbsp;Corporation</td><td>51118170</td><td></td></tr><tr><td>Colloid gold (40 nm) solution&amp;nbsp;</td><td>REGA biotechnology Inc.</td><td></td><td>40-50 nm is appropriate for immunostrip</td></tr><tr><td>Bovine serum albumin</td><td>Gibco</td><td>15561-020</td><td></td></tr><tr><td>Rapid test immno-strip printer</td><td>REGA biotechnology Inc.</td><td>AGISMART RP-1000&amp;nbsp;</td><td>Only suited for small scale production of immunostrips for research and development purposes</td></tr><tr><td>Strip components (NC membranes, sample pads (#33 glass, S&amp;amp;S), conjugate pads (#16S, S&amp;amp;S) and absorbent pads (CF6, Whatman))</td><td>REGA biotechnology Inc.</td><td></td><td></td></tr><tr><td>Freund&amp;rsquo;s adjuvant and incomplete Freund&amp;rsquo;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5881, F5506</td><td>Used to produce water-in-oil emulsions of immunogens</td></tr><tr><td>Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED)&amp;nbsp;</td><td>Protech</td><td></td><td>Gel preparation for SDS-PAGE</td></tr><tr><td>Coomassie brilliant blue R-250</td><td>Bio-Rad</td><td>1610436</td><td>Protein staining in SDS-PAGE gels</td></tr><tr><td>Laemmli sample buffer and &amp;beta;-mercaptoethanol</td><td>Bio-Rad</td><td>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 ...

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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

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Bioengineering

Fabricating a UV-Vis and Raman Spectroscopy Immunoassay Platform

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, a large infrastructure of immunoassay instruments and materials can be found. For example, 96- and 384-well polystyrene plates are available commercially and have a standard design to accommodate ultraviolet-visible (UV-Vis) spectroscopy machines from various manufacturers. In addition, a wide variety of immunoglobulins, detection tags, and blocking agents for customized immunoassay designs such as enzyme-linked immunosorbent assays (ELISA) are available.
Despite the existing infrastructure, standard ELISA kits do not meet all research needs, requiring individualized immunoassay development, which can be expensive and time-consuming. For example, ELISA kits have low multiplexing (detection of more than one analyte at a time) capabilities as they usually depend on fluorescence or colorimetric methods for detection. Colorimetric and fluorescent-based analyses have limited multiplexing capabilities due to broad spectral peaks. In contrast, Raman spectroscopy-based methods have a much greater capability for multiplexing due to narrow emission peaks. Another advantage of Raman spectroscopy is that Raman reporters experience significantly less photobleaching than fluorescent tags1. Despite the advantages that Raman reporters have over fluorescent and colorimetric tags, protocols to fabricate Raman-based immunoassays are limited. The purpose of this paper is to provide a protocol to prepare functionalized probes to use in conjunction with polystyrene plates for direct detection of analytes by UV-Vis analysis and Raman spectroscopy. This protocol will allow researchers to take a do-it-yourself approach for future multi-analyte detection while capitalizing on pre-established infrastructure.

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

Immunoassays are used to detect proteins based on the presence of associated antibodies. Because of their extensive use in research and clinical settings, ...

Biochemistry

Use of Autometallography to Localize and Semi-Quantify Silver in Cetacean Tissues

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong antimicrobial effects. They also may be released into the environment and accumulate in the ocean. Therefore, AgNPs are the major source of Ag contamination, and public awareness of the environmental toxicity of Ag is increasing. Previous studies have demonstrated the bioaccumulation (in producers) and magnification (in consumers/predators) of Ag. Cetaceans, as the apex predators of ocean, may have been negatively affected by the Ag/Ag compounds. Although the concentrations of Ag/Ag compounds in cetacean tissues can be measured by inductively coupled plasma mass spectroscopy (ICP-MS), the use of ICP-MS is limited by its high capital cost and the requirement for tissue storage/preparation. Therefore, an autometallography (AMG) method with an image quantitative analysis by using formalin-fixed, paraffin-embedded (FFPE) tissue may be an adjuvant method to localize Ag distribution at the suborgan level and estimate the Ag concentration in cetacean tissues. The AMG positive signals are mainly brown to black granules of various sizes in the cytoplasm of proximal renal tubular epithelium, hepatocytes, and Kupffer cells. Occasionally, some amorphous golden yellow to brown AMG positive signals are noted in the lumen and basement membrane of some proximal renal tubules. The assay for estimating the Ag concentration is named the Cetacean Histological Ag Assay (CHAA), which is a regression model established by the data from image quantitative analysis of the AMG method and ICP-MS. The use of AMG with CHAA to localize and semi-quantify heavy metals provides a convenient methodology for spatio-temporal and cross-species studies.

A protocol is presented to localize Ag in cetacean liver and kidney tissues by autometallography. Furthermore, a new assay, named the cetacean histological Ag assay (CHAA) is developed to estimate the Ag concentrations in those tissues.

Silver nanoparticles (AgNPs) have been extensively used in commercial products, including textiles, cosmetics, and health care items, due to their strong ...

Environment

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Gold Immunostaining of Exosomal Sections: A Technique to Visualize Diagnostic Markers in Exosomal Lumen

Source: Jung, M. K., Mun, J. Y. <a href="https://www.jove.com/video/56482" target="_blank">Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy.</a> J. Vis. Exp. (2018)
In this video, we demonstrate gold immunostaining of biomarkers localized within the lumen region of ultrathin exosomal sections. This technique enables early diagnosis of diseases by visualization of target molecules within the exosomes secreted by the diseased cells.

Source: Jung, M. K., Mun, J. Y. Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy. J. Vis. Exp. (2018)
In this video, we ...