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).

Declaración de Ética: El estudio se realizó de acuerdo con las directrices internacionales y aprobado por el Comité de Cuidado y Uso de Animales Institucional (IACUC) de la Universidad Nacional de Chiayi, aprobación ID: 99022. El uso de la muestra de cetáceos fue permitido por el Consejo de Agricultura de Taiwán (Permiso de Investigación 100M-02.1-C-99). 1. Preparación de la muestra del músculo y SDS-PAGE Nota: Las muestras de músculo de...

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El uso de un péptido sintético conjugado con la proteína portadora es notablemente más eficaz en comparación con su proteína afín. Para una técnica basada en sándwich, debido a que el MAB se desarrolla utilizando epítopos con ubicaciones relativas conocidas, los dos anticuerpos monoclonales en este estudio no pueden interferir con la interacción de cada uno con el epítopo del antígeno diana. Además, la reactividad entre la proteína nativa y el anticuerpo de ratones inmunizados con el conjugado de péptido...

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.

Características de anticuerpos monoclonales Hemos desarrollado dos IgG 1 mAb (CGF5H9 y CSF1H13) que reconocen dos péptidos sintéticos (MKASEDLKKHGNTVLC y AIIHVLHSRHPAEFGC), respectivamente, de los cetáceos Mb, y éstos se utilizaron para construir una tira de prueba inmunocromatográfica de oro coloidal de tipo sándwich para la detección rápida de Mb de cetáceos. Figura 2 muestra que CGF5H9 detecta cetáceos y otros mamíferos como una sol...

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.

Desarrollo de una tira de prueba inmunocromatográfica basada en el oro coloidal para la detección de Cetáceos mioglobina

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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