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 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.
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).
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.
2. Peptide Synthesis and Monoclonal Antibody Production
3. Western Blot
4. Dot Blot
5. Indirect ELISA
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.
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 (< 20% Relative Humidity) for prolonged storage life (> 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.
8. Cross-reactivity Test
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 approximate 17 kDa. The common minke whale (Balaenoptera acutorostrata) shows a comparatively fainter band than the bands of other cetaceans. Bands are absent for tuna, chickens and seals, whereas a band at about 50 kDa is observed for pigs. Although there are multiple nonspecific bands for pigs and tuna, CSF1H13 is highly specific because it reacts only with cetaceans and seals as a band at a predicted molecular weight of approximate 17 kDa. Minke whale only shows a strong signal at 1:10 (data not shown) with no signal observed at 1:25. Figure 2 shows identical results in dot blot.
Figure 3A shows that CGF5H9 demonstrates positive signal for cetaceans, rabbits, dogs, goats, and cows (OD value > 3.0); weak positive signal for pigs (OD value = 1.5); negative signal for seals, chickens and tuna. Figure 3B shows that CSF1H13 expresses high affinity towards cetaceans and seals (OD value > 3.0). Both CSF1H13 and CGF5H9 can react strongly with all four cetacean species. These four species are from different families (minke whale: Balaenopteridae; bottlenose dolphin: Delphinidae; dwarf sperm whale: Kogiidae; finless porpoise: Phocoenidae), indicating the broad reactivity to diverse cetacean species.
For strip construction, CGF5H9, which recognizes the Mbs of cetaceans and other mammals, is colloid gold-labeled and used as the detection antibody to bind myoglobin, and CSF1H13 is coated on the test line only to capture the Mbs of cetaceans and seals. Therefore the test line is designed to show a positive signal when both mAbs detect cetacean Mb. Because the Mb from non-cetacean animals can only be detected by one of these two mAbs, the test line shows a negative result when muscle samples from other animals are tested. The control line always shows a positive result because rabbit anti-mouse IgG binds colloid gold-labeled CGF5H9. A failed result in the control line indicates that the quality of the materials on the strip is poor.
Strip test
Figure 4 shows the signal bands at both test line and control line when cetacean muscle samples are used. When the sample is not from cetaceans, there is only a single band at the control line with the absence of band at the test line. A successful result can be observed directly in 5-10 min after homogenizing 0.03 g of muscle with 10 ml PBS containing 0.1% BSA using a plastic or bamboo stick and soaking the strip into the mixture. After testing 15 cetacean species and eight non-cetacean species in triplicate, specificity (the percentage of non-cetacean samples correctly identified) and sensitivity (the percentage of cetacean samples correctly identified) are both 100%.
Figure 1. Design of the immune strip. All the components are carefully layered on to the plastic backing card so that they overlap. This allows the reagents and sample flow up through the membrane and to the absorbent pad. T: test zone. C: control zone. This figure has been reproduced from Lo, C. et al. Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health. PLoS ONE 8, e60704 (2013). doi:10.1371/journal.pone.0060704. Please click here to view a larger version of this figure.
Figure 2. Western blot and dot blot analysis using the hybridoma supernatants. (A) CGF5H9, (B) CSF1H13. Both hybridoma supernatants can detect cetaceans as a single stained band at a predicted molecular weight of approximate 17 kDa. MW: minke whale, BND: bottlenose dolphin, DSW: dwarf sperm whale, FP: finless porpoise, PKW: pygmy killer whale, N: PBS (negative control). This figure has been reproduced from Lo, C. et al. Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health. PLoS ONE 8, e60704 (2013). doi:10.1371/journal.pone.0060704. Please click here to view a larger version of this figure.
Figure 3. Indirect ELISA of muscle extracts of different species using purified mAbs. (A) CGF5H9, (B) CSF1H13. Only cetaceans can produce strong positive signals in both mAbs. This figure has been reproduced from Lo, C. et al. Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health. PLoS ONE 8, e60704 (2013). doi:10.1371/journal.pone.0060704. Please click here to view a larger version of this figure.
Figure 4. Specificity of the immune colloidal gold strip. T: test line. C: control line. Only when cetacean muscle samples are used, successful results (signal bands at both test line and control line) can be observed. (A) Non-cetacean samples: 1: Cow. 2: Goat. 3: Pig. 4: Dog. 5: Rabbit. 6: Tuna. 7: Chicken. 8: Harbor seal (Phoca vitulina). (B) Cetacean samples: 1: Common minke whale (Balaenoptera acutorostrata). 2: Omura's whale (Balaenoptera omurai). 3: Bottlenose dolphin (Tursiops aduncus). 4: Bottlenose dolphin (T. truncatus). 5: Fraser's dolphin (Lagenodelphis hosei). 6: Indo-Pacific humpback dolphin (Sousa chinensis). 7: Risso's dolphin (Grampus griseus). 8: Pantropical spotted dolphin (Stenella attenuata). 9: Rough-toothed dolphin (Steno bredanensis). 10: Pygmy killer whale (Feresa attenuata). 11: Short-finned pilot whale (Globicephala macrorhynchus). 12: Melon-headed whale (Peponocephala electra). 13: Dwarf sperm whale (Kogia sima). 14: Pygmy sperm whale (K. breviceps). 15: Finless porpoise (Neophocaena phocaenoides). This figure has been reproduced from Lo, C. et al. Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health. PLoS ONE 8, e60704 (2013). doi:10.1371/journal.pone.0060704. Please click here to view a larger version of this figure.
Cetacean species | Non-cetacean species |
Pygmy sperm whale (Kogia breviceps) | Harbor seal (Phoca vitulina) |
Dwarf sperm whale (Kogia sima) | Dog (Canis lupus familiaris) |
Short-finned pilot whale (Globicephala macrorhynchus) | Rabbit (Oryctolagus cuniculus) |
Melon-headed whale (Peponocephala electra) | Pig (Sus scrofa) |
Pygmy killer whale (Feresa attenuata) | Goat (Capra hircus) |
Pantropical spotted dolphin (Stenella attenuata) | Cattle (Bos Taurus) |
Bottlenose dolphin (Tursiops truncatus) | Chicken (Gallus gallus) |
Bottlenose dolphin (Tursiops aduncus) | Yellowfin tuna (Thunnus albacares) |
Fraser’s dolphin (Lagenodelphis hosei) | |
Indo-Pacific humpback dolphin (Sousa chinensis) | |
Rough-toothed dolphin (Steno bredanensis) | |
Risso’s dolphin (Grampus griseus) | |
Finless porpoise (Neophocaena phocaenoides) | |
Common minke whale (Balaenoptera acutorostrata) | |
Omura’s whale (Balaenoptera omurai) |
Table 1. The species from which muscle was collected and tested in this study. Species include tuna, chicken, seal, 5 species of terrestrial mammals, and 15 species of cetaceans (4 families).
Species | Accession no. |
Common minke whale (Balaenoptera acutorostrata) | P02179 |
Pygmy Bryde's whale (Balaenoptera edeni) | Q0KIY2 |
Humpback whale (Megaptera novaeangliae) | P02178 |
Gray whale (Eschrichtius robustus) | P02177 |
Sperm whale (Physeter macrocephalus) | P02185 |
Pygmy sperm whale (Kogia breviceps) | Q0KIY5 |
Dwarf sperm whale (Kogia sima) | P02184 |
Short-beaked common dolphin (Delphinus delphis) | P68276 |
Long-finned pilot whale (Globicephala melas) | P02174 |
Killer whale (Orcinus orca) | P02173 |
Melon-headed whale (Peponocephala electra) | Q0KIY3 |
Pantropical spotted dolphin (Stenella attenuata) | Q0KIY6 |
Bottlenose dolphin (Tursiops truncatus) | P68279 |
Harbor porpoise (Phocoena phocoena) | P68278 |
Amazon river dolphin (Inia geoffrensis) | P02181 |
Longman's beaked whale (Indopacetus pacificus) | Q0KIY9 |
Hubbs' beaked whale (Mesoplodon carlhubbsi) | P02183 |
Cuvier's beaked whale (Ziphius cavirostris) | P02182 |
Harbor seal (Phoca vitulina) | P68080 |
Cattle (Bos Taurus) | P02192 |
Goat (Capra hircus) | B7U9B5.3 |
Horse (Equus caballus) | P68082 |
Pig (Sus scrofa) | P02189 |
Dog (Canis lupus familiaris) | P63113 |
Chicken (Gallus gallus) | P02197 |
Ostrich (Struthio camelus) | P85077 |
Yellowfin tuna (Thunnus albacares) | P02205 |
Table 2. Myoglobin sequences used in this study with respective GenBank accession numbers. Species include tuna, chicken, ostrich, domestic mammals, seal and 18 species of cetaceans (7 families). This table has been reproduced from Lo, C. et al. Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health. PLoS ONE 8, e60704 (2013). doi:10.1371/journal.pone.0060704.
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'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 protein and the antibody produced from the native protein19. The use of synthetic peptide conjugates is therefore recommended for effective immunization procedures and generation of appropriate anti-peptide mAb.
The structure of a protein mainly involves the sequence of amino acids in the polypeptide chain. Each amino acid has its side-chain leading to specific properties, and slightly changing the amino acid sequence results in structure changes. Because peptides with a length of 10-20 amino acids are ideal for antibody preparation, the length of the synthetic peptides (immunogen) at the C-terminal region was increased to ensure that the core antigenic region would be recognized. Therefore, the amino acid residues among Mbs of various animals resulting in different epitope structures could be efficiently differentiated. For example, Figure 3A shows the mentioned peptide design contributes to CGF5H9 reacting strongly with cetaceans but negatively with seals. Another example is the distinct affinities toward chickens and dogs of CGF5H9 although the chicken has an identical sequence to that of the dog in the core antigenic site 2. This indicates that the sequence difference in the outer region could lead to the structure change and thus variable binding affinity between antigen and antibody.
Western blot, dot blot, and indirect ELISA were used in our method for screening suitable mAbs. Western blot is widely used to detect specific proteins in tissue extract or homogenate. In this technique, gel electrophoresis is used to separate denatured proteins by the length of the polypeptide. Therefore, it is possible to confirm if the signal indicates the predicted protein molecular weight. However, the detection result (positive or negative, strong or weak signal) may not have represented the real situation of antigen-antibody binding because denatured proteins are used. Consequently, dot blot can be used for second-stage screening. Dot blot is a technique for detecting proteins. It represents a simplification of western blot method. In dot blot, a mixture containing the molecule to be detected is applied directly on a membrane as a dot. This differs from a western blot because protein samples are not denatured. Note that this technique offers no information on the size of the target biomolecule, and a single dot will appear if two molecules of different sizes are detected. Finally, indirect ELISA is used for a ligand-binding assay in order to generate a signal that can be properly quantified. It provides more information of mAb characteristics and thereby facilitates the strip construction.
Concentrations of Mb in the muscle are variable depending on the collection location. For example, swimming muscles (axial muscles) in cetaceans have a significantly higher content of Mb compared with non-swimming muscles, and samples from young cetaceans would have lower Mb concentration because the Mb concentration increases throughout an animal's life20. Initially, CSF1H13, which only captures the Mb of cetaceans and seals, was intended to be colloid gold-labeled and be detecting antibody, and CGF5H9, which recognizes the Mb of many species, would be capture antibody on the test line. We hypothesized that the detecting antibody should be more specific and the capture antibody should be more general. However, a weak positive signal was found on the test line when cetacean samples with low Mb concentrations were used (data not shown). The problem was resolved when the positions of the two mAbs were reversed as described in the representative results. A good signal was even shown for a newborn cetacean (a stranded Omura's whale) (Figure 4). It is unclear whether the characteristics and concentration of mAbs contribute to this phenomenon.
In this study, frozen-thawed muscle samples homogenized with PBS were used on the strip test. Other sample conditions and preparation methods may affect the result. For example, salt-soluble protein such as Mb should be extracted using PBS rather than pure water. Otherwise, the extraction may be inadequate, which could lead to an aberrant result. The appropriate extraction buffer: meat sample ratio is partly responsible for the successful interpretation of the strip result. Large amount (0.3 g in 1 ml buffer) of control samples (e.g., domestic animals) could cause positive result and blurred background. However, the ratio used in this study (1: 0.03) produced correct results. Only fresh muscle samples can be used for this strip test. Protein could be hydrolyzed or denatured after certain treatments (such as curing by soy sauce and boiling), which could cause positive results not only for cetacean muscle samples but also samples from other animals (data not shown). Therefore, it is suggested that variable sample sources and different construction design plans should be used during strip development.
In conclusion, this protocol describes the development of two mAbs strongly reactive to the Mb of cetaceans, and these mAbs are applied on a quick test strip to differentiate the Mb of cetaceans from seal and other animals. Although reliable PCR-based DNA analysis for the identification of cetacean meat is available12, it is labor intensive and time consuming. The quick test strip is a dependable and rapid technique that can be used in the field to identify cetacean meats, which is highly desirable for regulatory agencies21. It is likely that the strip can be developed for detecting specific Mbs from animals such as horses or pigs.
The authors have nothing to disclose.
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).
Phosphate buffered saline | AMRESCO | J373 | |
Protein G HP SpinTrap | GE Healthcare | 28-9031-34 | spin column containing Protein G Sepharose |
IsoStrip Mouse Monoclonal Antibody Isotyping Kit | Roche | 11493027001 | Isotyping Strips, precoated with subclass- and light-chain-specific anti-mouse-Ig antibodies |
Mini Trans-Blot | Bio-Rad | 170-3935 | |
Nitrocellulose membrane | Whatman | Z613630 | |
Antibody blocker solution | LTK BioLaboratories | To minimize nonspecific binding interactions of nonspecific IgG in the samples | |
BCIP/NBT phosphatase substrate | KPL | 50-81-00 | |
Protein Detector HRP Microwell Kit, Anti-Mouse | KPL | 54-62-18 | |
Nunc Immunoplate MaxiSorp ELISA plate | Thermo Fisher Scientific | EW-01928-08 | |
Multiskan EX ELISA reader | Thermo Electron Corporation | 51118170 | |
Colloid gold (40 nm) solution | REGA biotechnology Inc. | 40-50 nm is appropriate for immunostrip | |
Bovine serum albumin | Gibco | 15561-020 | |
Rapid test immno-strip printer | REGA biotechnology Inc. | AGISMART RP-1000 | Only suited for small scale production of immunostrips for research and development purposes |
Strip components (NC membranes, sample pads (#33 glass, S&S), conjugate pads (#16S, S&S) and absorbent pads (CF6, Whatman)) | REGA biotechnology Inc. | ||
Freund’s adjuvant and incomplete Freund’s adjuvant | Sigma-Aldrich | F5881, F5506 | Used to produce water-in-oil emulsions of immunogens |
Acrylamide, gel buffer, ammonium persulfate (APS), tetramethylethylenediamine (TEMED) | Protech | Gel preparation for SDS-PAGE | |
Coomassie brilliant blue R-250 | Bio-Rad | 1610436 | Protein staining in SDS-PAGE gels |
Laemmli sample buffer and β-mercaptoethanol | Bio-Rad | 1610737, 1610710 | Dilute protein samples before loading on SDS-PAGE gels |