Name: development of a colloidal gold-based immunochromatographic test strip for detection of cetacean myoglobin
Uploaded: 2016-07-13T13:00:00
Description: Watch this Scientific Journal Video about Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin at JoVE.com

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 terres...

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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).

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	<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 ...

The overall goal of this colloidal gold immunochromatographic test strip is to differentiate the myoglobin of cetaceans from that of seals and other animals. This method can provide rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. The main advantage of this technique is that a successful result can be observed directly in five to ten minutes after homogenizing 03 gram of muscle, with outstanding specificity and sensitivity.So this method can provide rapid detection of cetacean meat in the field. It can also be applied to other meat such as horse meat, or pork. To design synthetic peptides for raising monoclonal antibodies with reactivity toward the myoglobin of cetaceans, the amino acid sequences of myoglobin antigenic reactive regions from various animals are analyzed.Begin by retrieving from GenBank the amino acid sequences of myoglobin from tuna, chicken, ostrich, domestic mammals, seal, and 18 species of cetaceans. Next, align the sequences using the proper software. 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, are you building a DNA or Protein Sequence alignment? Click the button labeled Protein, 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.Select Alignment Align by ClustalW from the main menu. Select BLOSUM as the protein weight matrix and then click the OK button. The third step is to analyze the sequence alignment.Focus on five antigenic reactive sites and find the fragment conserved among cetaceans. An asterisk symbolizes consensus in the alignment and indicates a position that has a single, fully-conserved residue. Two conserved fragments in cetaceans were found.Candidate sequence fragments are then synthesized and used to raise monoclonal antibodies. Begin this procedure by determining the optimal pH for the colloidal gold solution defined as the minimum pH that keeps the monoclonal antibody and colloidal gold solution mixture red in color for two hours. Add 0.15 micrograms of purified detecting monoclonal antibody to 100 microliters of colloidal gold solution with pH values varying from five to nine.For the purposes of this demonstration, pH six and pH eight are tested. pH eight was considered the optimal pH by this optimization step. Next, determine the optimal concentration of the monoclonal antibody by adding various amounts of purified detecting monoclonal antibody to 100 microliters of colloidal gold solution at pH eight.The optimum concentration, which is six micrograms per milliliter in this study, will keep the color of the mixture red with no black precipitate. Based on the results from the optimization steps, add 60 micrograms of purified detecting monoclonal antibody drop-wise to 10mL of colloidal gold solution. Emulsify the mixture gently at room temperature for ten minutes.Add two milliliters of a 5%BSA solution in PBS to the mixture and emulsify gently at room temperature for 15 minutes to reduce background interference. Centrifuge the mixture at 10, 000 times g for 30 minutes at four degrees Celsius. After centrifugation, carefully remove the supernatant with unconjugated antibody and suspend the resulting pellet in 4mL of PBS-T containing 1%BSA and 0.1%Tween-20.Repeat the centrifugation and suspension once or twice. Suspend the final precipitates in 1mL of PBS-T and store at four degrees Celsius until use. A graphic of the immune strip design is shown here.The dimensions of the cardboard, pads, and nitrocellulose membrane are listed in the protocol text. To prolong storage life, the immune strips should be prepared and assembled in a low-humidity laboratory environmental condition. Use a micropipette to add the colloid gold-labeled monoclonal antibody solution to the conjugate pad to saturate it and then dry the pad at 37 degrees Celsius for one hour before assembling.Once the conjugate pad is dry, use double-sided tape to first paste the nitrocellulose membrane onto the cardboard. Using an immunostrip printer, distribute the specific antigen capturing monoclonal antibody and the rapid anti-mouse IGG on the test zone and the control zone, respectively, on the nitrocellulose membrane. Maintain a distance of greater than five millimeters between the two zones.Then paste the conjugate pad on one side of the nitrocellulose membrane, and the absorbent pad on the other side, overlapping the pads on each side by about two millimeters. Place the sample pad over the conjugate pad by two millimeters and paste it on the cardboard. Use a paper cutter to create six-millimeter-wide strips.Pack the strips in an aluminum foil bag with dessicant and store at four degrees Celsius until use. To prepare a sample for the cross-reactivity test, add 1mL of PBS containing 0.1%BSA to 0.03 grams of raw muscle. And use a bamboo stick or a grinding rod to homogenize the sample.Hold the immune strip by the end opposite to the test area and dip the sample pad part into the specimen for five to ten minutes. Observe the result directly. Test various muscle samples in triplicate.The T test line is designed to show a positive signal when both monoclonal antibodies detect cetacean myoglobin. Because the myoglobin from non-cetacean animals can only be detected by one of these two monoclonal antibodies, the test line shows a negative result when muscle samples from other animals are tested. The C control line should always show a positive result because rapid anti-mouse IGG binds the colloid gold-labeled detection antibody.A failed result in the Control line indicates that the quality of the materials on the strip is poor. Western blood analysis using hybridoma supernatants shows that the CGF5H9 monoclonal antibody detects cetaceans and other mammals as a single stained band at a predicted molecular weight of approximately 17kDa. The common Minke whale shows a comparatively fainter band than the bands of other cetaceans.Bands are absent for tuna, seal, and chicken. Whereas a band at about 50kDa is observed for pig. Identical results are obtained by dot blood analysis.Although Western blood analysis using the CSF1H13 monoclonal antibody shows multiple nonspecific bands for pig and tuna, CSF1H13 is highly specific because it reacts only with cetaceans. And seal has a band at a predicted molecular weight of approximately 17kDa. Identical results are obtained by dot blood analysis.Results from Indirect ELISA of muscle extracts of different species using purified CGF5H9 demonstrate positive signals for cow, goat, dog, rabbit and cetaceans, a weak positive signal for pig, and negative signals for tuna, chicken, and seal. CSF1H13 shows high affinity towards cetaceans and seals. Both monoclonal antibodies can react strongly with all four cetacean species which are from different families, indicating their broad reactivity to diverse cetacean species.The specificity of the immune colloidal gold strip is shown by these representative results. The use of non-cetacean muscle samples results in only a single band at the control line. However, when cetacean muscle samples are used, the signal bands are present at both the test line and control line.Once mastered, the preparation of labeled antibodies and this construction of strips can be done in four hours if it's performed properly. When attempting this procedure, it's important to remember to design the peptide carefully. Use several methods for screening suitable monoclonal antibodies.And use a high quality of colloidal gold to prepare labeled antibodies. Following these procedures, other methods like PCR-based DNA analysis can be performed in order to answer additional questions, like the cetacean species and from where it came. The CSF1H13 monoclonal antibody which is strong and exhibits an affinity towards only marine mammals can be employed in various formats of ELISA that offer many other opportunities for studying the biological, physiological and chemical aspects of myoglobin in marine mammals.After watching this video, you should have a good understanding of how to develop specific monoclonal antibodies and subsequently apply them over quick test strip.

Research

JoVE Journal

Biology

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 ...

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 ...

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) ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

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 ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

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 ...

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 ...

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 ...

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 ...