title: Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin.
abstract: 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.
authors: Department of Veterinary Medicine, National Chiayi University.
authors: Kun-Wei
authors: KW
authors: Chan
authors: Department of Veterinary Medicine, National Chiayi University.
authors: Chieh
authors: C
authors: Lo
authors: Department of Microbiology and Immunology/Graduate Institute of Biomedical and Biopharmaceutical Sciences, National Chiayi University.
authors: Chi-Shih
authors: CS
authors: Chu
authors: Department of Microbiology and Immunology/Graduate Institute of Biomedical and Biopharmaceutical Sciences, National Chiayi University.
authors: Li-Te
authors: LT
authors: Chin
authors: Department of Veterinary Medicine, National Chiayi University.
authors: Yu-Ting
authors: YT
authors: Wang
authors: Department of Veterinary Medicine, National Chiayi University; jackywc@gmail.com.
authors: Wei-Cheng
authors: WC
authors: Yang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002591:Cetacea; D060326:Chemistry Techniques, Synthetic; D002846:Chromatography, Affinity / Q000379:methods*; D017979:Gold Colloid / Q000737:chemistry*; D009211:Myoglobin / Q000032:analysis*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D017979:Gold Colloid; D009211:Myoglobin