New Immunoassay Method For Malachite Green And Crystal Violet

In view of the food safety hazards caused by the illegal use of malachite green and crystal violet in aquaculture,two immunoassay methods,fluorescence quenching immunoaffinity gel detection column(FQ-ITC)analysis method and fluorescent labeled magnetic separation competitive immunoassay(FI-MSI),were established for simultaneous detection of malachite green(MG)and crystal violet(CV)residues.A visual fluorescence quenching immunoaffinity test column(FQ-ITC)assay was developed based on fluorescence resonance energy transfer from the fluorescence donor(quantum dot)to the fluorescence acceptor(MG or CV)and the specific identification between the analytes and their polyclonal antibodies.On the basis of optimizing the preparation conditions and testing conditions of FQ-ITC,the FQ-ITC assay was established and the specificity of the assay was analyzed.The sample matrix effect of aquatic products was further analyzed and the sample preparation method and matrix elimination method were optimized.The proposed FQ-ITC assay can be successfully applied to the simultaneous detection of MG or CV residues in aquatic products such as grass carp,striped bass,carp,and shrimp.The process can be completed within 10 minutes,and the results can be visually judged with a visual limit of detection of 2 ?g/kg.Fluorescent labeled magnetic separation competitive immunoassay(FI-MSI)utilizes the principle of competitive binding of analytes and sensing probes to signal probes.The malachite green and crystal violet were detected rapidly by the change of fluorescence intensity of the reaction system.Signal probes and induction probes were prepared by the activated ester method.The amount of MG and CV standards and signal probes was 10 ?L.The amount of sensing probes was 30 ?L.The total volume of the reaction system was 200 p.L.The incubation time was 70 min.The fluorescence detection magnetic separation immunoassay method has a limit of detection of 0.012?g/L with a linear range of 0.1-1000 ?g/L.Structural analogues of analytes and drugs commonly used in aquatic products were selected for cross-over experiments.The results show that the FI-MSI has good specificity.On the basis of optimizing the establishment of sample preparation methods and matrix elimination methods,the assay can be used for the quantitative analysis of four kinds of aquatic products such as grass carp,striped bass,carp,and shrimp.The limit of detection of the sample was 0.144 pg/kg,the recovery rate of adding MG was 80.0%-98.4%,and the recovery rate of CV was 80.0%-102.0%.The established methods were compared with liquid chromatography tandem mass spectrometry(LC-MS/MS)and commercial ELISA kits,respectively,to verify the validity of the established analytical methods.Both new immunoassays can be applied to the rapid simultaneous detection of malachite green and crystal violet in aquatic products.