Establishment Of AlphaLISA For Detection Of Veterinary Drug Residues In Animal Food

Animal food is a common and most important class of food in human diet.The raw materials of this kind of food are edible meat and tissues of various animals,After different methods of processing,become different types of meat food.Common meat includes pork,beef,fish and so on.These foods are rich in nutrients,high absorption rate and delicious,can be provided to the human body essential nitrogen acid,fatty acids,inorganic salts,vitamins and other substances.In recent years,the demand for meat products has increased and prices have continued to grow.However,fish susceptible to bacterial invasion in the growth process,aquaculture farmers use malachite green to treat parasites,fungi or bacterial infections.The growth period of livestock is longer and lean meat rate is not high.Some of the farmer on the use of malachite green,zearalcohol,β-agonists and other drugs,in order to improve lean meat,reduce fat content,shorten the slaughter time.These drugs are usually added to the feed,or in the form of ear burial to the animals,easily lead to drug residues.Malachite green,especially its metabolites in aquatic animals,there are significant high residue and high toxicity,can produce teratogenic,carcinogenic,mutagenic and other side effects;ZAL can cause human sex hormone dysfunction and affect the normal development of secondary sexual characteristics,induced by external conditions,may cause cancer.But also by drinking water and food caused by secondary pollution and environmental pollution;β-agonist easy to accumulate in the animalorgans,and through the food chain into the body when with more than 5-10 times the dose of the therapeutic dose for livestock breeding.If excessive intake may lead to mutagenesis,carcinogenesis or teratogenesis,and even damage the liver and kidney.In order to protect the health of consumers,China,the European Union and other countries prohibit use of veterinary drug.The evaluation of veterinary drugs is no longer limited to its economic benefits to meat products,but more emphasis on environmental and health benefits.Therefore,the detection of meat veterinary drug residues has a very important significance.Methods for veterinary drug testing are HPLC,MS / MS-HPLC,ELISA,etc.,but these methods have some shortcomings(for example,can not be large-scale screening detection,high cost).Thus,a variety of veterinary drug testing,low detection limit,large-scale screening detection has become the development of demand,which can not only guarantee the quality and safety of meat,but also can reduce the human,financial and material.In this paper,an efficient,rapid and sensitive screening method was established for veterinary drug residues detection in animal food.Part ⅠQuantitative determination of Malachite green using amplified luminescent proximity homogeneous assay in aquatic product Objective: A new method was established for analyzing Malachite green(MG)by amplified luminescent proximity homogeneous assay(Alpha LISA).Methods: Analyte,botinylated MG-BSA and MG antibody were added to 384-well microplates,then added donor beads and acceptor beads,and then detected by Alpha LISA method.Results: TheMG-Alpha LISA with Leucomalachite green and Leucomalachite crystal violet were not cross-reactivity,respectively,the sensitivity was 0.42ng/m L.The recoveries of the determination for MG in two kinds of fish samples were 81.0%–105.0% and 84.0%–90.8%,respectively.The RSD of intra–day and inter–assay were <9.5% and <16.0%,respectively.Conclusion: The MG-Alpha LISA had the characteristics of excellent specificity and sensitivity,and stability.It could be widely used in rapid and simple screening for MG contamination in fish.Part Ⅱ Determination of Zearalanol and Its Analog Zearalanone in Muscle Tissues by Amplified Luminescent Proximity Homogeneous Assay(Alpha LISA)Objective: A homogenous light-induced chemiluminescence immunoassay(Alpha LISA)method was established for the determination of residues of Zearalanol and its analog Zearalanone in muscle tissue samples.Methods:Pork and bovine muscles were used as raw material.The samples were extracted with sodium acetate and β-glucuronidase/Sulfatase.The extracts was purified by HLB,and then detected by Alpha LISA method.Results:The method showed a linear relationship in the range of 0.01-4 ng/m L(R2 > 0.99);the sensitivity of the assay was 0.066 ng/m L.Crossreactivity rate of Zearalanol was 100% and Zearalanone was 82.1%,and other compounds were not more than 40%.The average recovery rates of Zearalanol at spiked levels of 1-4 ng/m L were 96.3% to 105.0%,and91.7% to 100.5% for pork and bovine muscles;the RSD<17.5%.Conclusion: These results indicated that the method is suitable applied in the analysis of Zearalanol and Zearalanone in muscle tissues.Part Ⅲ Quantitative Determination of β-Agonists Using the Amplified Luminescent Proximity Homogeneous Assay in Pork Objective: A amplified luminescent proximity homogeneous assay(Alpha LISA)was developed for determination of four kinds of β-agonists(clenbuterol,salbutamol,brombuterol,terbutaline)in pork.Methods: pork was used as raw material.The analytes were stepwise extracted with ammonium acetate and β-glucuronidase / sulfatase,the extract was columned and concentrated to dryness,redisolved in buffer.Sample with botinylated antigen,antibody,donor beads and acceptor beads were reacted,and detected by Alpha LISA apparatus.Results: As a result,there was a good linear relationshipg in the range of 0.1-500 ng/m L,with correlation coefficient larger than 0.98.The cross-reactivity of the clenbuterol,brombuterol,terbutaline was 143.3%,179.2% and 95.6%,respectively,the sensitivity was 0.03-0.08 ng/m L.The average recoveries of four compounds at spiked levels of 0.5,10,20 μg/kg were in range of 82.5%-115.9%,the RSD<12.0%.Conclusion: The method is simple,rapid and accurate,and is suitable for the detection and screening of β-agonists in pork.