Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies.
To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.
Ministry of the Flemish Community, Agricultural Research Centre Ghent (CLO), Department of Animal Product Quality and Transformation Technology (DVK), Brusselsesteenweg 370, B-9090 Melle, Belgium. e.vancoillie@clo.fgov.be
Els
E
Van Coillie
Jan
J
De Block
Wim
W
Reybroeck
D000818:Animals; D002645:Chickens / Q000276:immunology; D004530:Egg Yolk / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D024841:Fluoroquinolones; D007136:Immunoglobulins; D008892:Milk / Q000737:chemistry*; D011807:Quinolizines / Q000032:analysis* / Q000276:immunology; D012680:Sensitivity and Specificity
D024841:Fluoroquinolones; C016512:IgY; D007136:Immunoglobulins; D011807:Quinolizines; C012976:flumequine