Nitrofurantoin And Furazolidone Residues In Food Enzyme-linked Immunoassay

With the development of society and the improvement of life level,the need for the animal food is increasing rapidly.Therefore,modern animal husbandry is developed very rapidly in recent years.To make more money,many farmers use some drugs in animal husbandry.Nitrofurans as a group of synthetic antibiotics are used widely to treat animal diseases or as feed additives.But long-term research showed that the nitrofurans and their metabolites showed carcinogenic,abnormal and mutagenic characteristics,so the illegal use of these drugs in animal husbandry is inevitable to result in the presence of residues in edible tissues and can have potential health risks to human.Therefore,Nitrofurans are controlled strictly used in aquiculture and poultry in many countries,such as Europe,America,China and Japan. So the safety issue of food has been highly concerned in the world.To ensure the safety of food for consumers,many countries have established analytical techniques to detect residues of drugs.The traditionally instrumental analyses such as HPLC,LC-UV,LC-MS and LC-MS-MS are the most widely used methods to detect residues of drugs in food and food products.These methods are sensitive,accurate and highly specific,but require expensive equipment,large volume of solvents,derivative treatment,and time-consuming sample clean-up process. Therefore,they are not suitable to be used as routing screen and field detection methods for drugs.To detect residue of drugs in food and food products,ELISA is a low cost,high volume screening methods.So we can combine ELISA methods with instrumental analyses to ensure efficient and cost-effective use of resources.The aim of this research is to establish the ELISA techniques to detect the residues of nitrofurantoin and furazolidone and their metabolites AOZ and AHD.To develop a rapid and convenient detection method to measure the residue of nitrofurantoin and AHD,we designed several immunogens via different methods on the basis of the structures of nitrofurantoin and AHD and prepared polyclonal antibodies to develop an immunoassay in this study.At the same time,we prepared coating antigen by the same methods as the preparation of the immunogen.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect nitrofurantoin and AHD(in the form of NPAHD)at one time.The immunoassay based on the antibody for nitrofurantoin showed excellent specificity and sensitivity with IC₅₀of 3.2ng/mL and no detectable cross reaction with most related species and compounds except with CPAHD and NPAHD.The limit of detection,defined as IC₁₀, was 0.2ng/mL.Considering that nitrofurantoin is often used illegally to feed animals through drinking water,we measured the residue of nitrofurantoin in water spiked by the drug.The recovery rates are in the range of 88-103%for inter-assay and 90-103% for intra-assay.The CVs are in the range of 3.1-11.4%for inter-assay and 2.7-6.2% for intra-assay.The immunoassay based on the antibody for AHD showed excellent specificity and sensitivity with IC₅₀(equivalent underivatised AHD)is 10.6ng/mL and no detectable cross reaction with nitrofurans and other nitrofuran metabolites except with CPAHD and nitrofurantoin.The limit of detection was 0.05ng/mL(equivalent underivatised AHD).For the residue of AHD in swine liver,the recovery rates are in the range of 80-96%for inter-assay and 91-104%for intra-assay.The CVs are in the range of 2.4-17.7%for inter-assay and 3.7-14.6%for intra-assay.To develop an antibody that is specific to furazolidone and AOZ,we designed the immunogen and coating antigen by the similar methods as the preparation of the immunogen and coating antigen of nitrofurantoin and AHD.The antibody obtained via acid anhydride scheme which is detected through the coating antigen prepared by glutaricdialdehyde method is successful.It can detect furazolidone and AOZ(in the form of NPAOZ)at one time.For the residue of furazolidone,the immunoassay based on the antibody showed excellent specificity and sensitivity with IC₅₀of 2.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and NPAOZ.The limit of detection was below 0.2ng/mL.We measured the residue of furazolidone in water spiked by the drug.The recovery rates are in the range of 74-88%for inter-assay and 76-92%for intra-assay.The CVs are in the range of 3.8-14%for inter-assay and 3.4-9.2%for intra-assay.The immunoassay based on the antibody for AOZ showed excellent specificity and sensitivity with IC₅₀of 3.9ng/mL and no detectable cross reaction with most related species and compounds except with CPAOZ and furazolidone.The limit of detection was about 0.18ng/mL (equivalent underivatised AOZ).In conclusion,we have successfully prepared the immunogens and coating antigens for nitrofurantoin and furazolidone as well as their metabolites,and we developed immunoassays based on the antibodies prepared successfully.The indirect ELISA methods developed in this study can be used as screen methods to detect residues in foods and food products.