title: Design and characterization of a direct ELISA for the detection and quantification of leucomalachite green.
abstract: Malachite green (MG), a member of the N-methylated triphenylmethane class of dyes, has long been used to control fungal and protozoan infections in fish. MG is easily absorbed by fish during waterborne exposure and is rapidly metabolized into leucomalachite green (LMG), which is known for its long residence time in edible fish tissue. This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of LMG in fish tissue. This development includes a simple and versatile method for the conversion of LMG to monodesmethyl-LMG, which is then conjugated to bovine serum albumin (BSA) to produce an immunogenic material. Rabbit polyclonal antibodies are generated against this immunogen, purified and used to develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for the screening and quantification of LMG in fish tissue. The assay performed well, with a limit of detection (LOD) and limit of quantification (LOQ) of 0.1 and 0.3 ng g(-1) of fish tissue, respectively. The average extraction efficiency from a matrix of tilapia fillets was approximately 73% and the day-to-day reproducibility for these extractions in the assay was between 5 and 10%.
authors.affiliation: Food Directorate, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Gurmit
authors.initials: G
authors.lastname: Singh
authors.forename: Terence
authors.initials: T
authors.lastname: Koerner
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Gelinas
authors.forename: Michael
authors.initials: M
authors.lastname: Abbott
authors.forename: Beth
authors.initials: B
authors.lastname: Brady
authors.forename: Anne-Catherine
authors.initials: AC
authors.lastname: Huet
authors.forename: Caroline
authors.initials: C
authors.lastname: Charlier
authors.forename: Philippe
authors.initials: P
authors.lastname: Delahaut
authors.forename: Samuel Benrejeb
authors.initials: SB
authors.lastname: Godefroy
mesh_terms: D000818:Animals; D000890:Anti-Infective Agents / Q000032:analysis; D017756:Aquaculture / Q000331:legislation & jurisprudence; D002170:Canada; D004396:Coloring Agents / Q000032:analysis; D003429:Cross Reactions; D004350:Drug Residues / Q000032:analysis* / Q000592:standards; D004797:Enzyme-Linked Immunosorbent Assay; D005506:Food Contamination; D005513:Food Inspection / Q000379:methods*; D006801:Humans; D007881:Legislation, Food; D057230:Limit of Detection; D015203:Reproducibility of Results; D012394:Rosaniline Dyes / Q000032:analysis* / Q000737:chemistry; D017747:Seafood / Q000032:analysis* / Q000592:standards; D052616:Solid Phase Extraction; D017210:Tilapia; D019155:Veterinary Drugs / Q000032:analysis; D014874:Water Pollutants, Chemical / Q000032:analysis
chemical_list: D000890:Anti-Infective Agents; D004396:Coloring Agents; D012394:Rosaniline Dyes; D019155:Veterinary Drugs; D014874:Water Pollutants, Chemical; C005095:malachite green; C015320:leucomalachite green