Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

Summary

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.

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.

Introduction

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 appearan....

Protocol

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 individual....

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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