Immunoassay Development For Six Chemicals Residues In Food

The scientific and rational use of chemical drugs is conducive to improving the yield and quality of agricultural and livestock products,safeguarding human health,and playing a pivotal role in food safety.Improper use of chemical drugs leads to excessive pesticide and veterinary drugs residues in food,causing harm to the ecological environment and national health.The six chemical drugs of grave concern were used as research objects,including four pesticides（diamide,prometryn,imazalil,pyrimethanil）and two veterinary drugs（dapsone,hyoscyamine）.Six specific and highly sensitive monoclonal antibodies against the drugs were prepared.Based on the antibodies,enzyme-linked immunosorbent assay and colloidal gold immunochromatography assay were developed and applied to determine the six chemicals residues in food.Based on the chemical structural characteristics of the research objects,haptens were designed and synthesized.Based on the core structure of cyclaniliprole,cyantraniliprole,chlorantraniliprole and tetrachlorantraniliprole,the benzamide was used as the spacer arm site,and the active carboxyl group was introduced to prepare diamide hapten.The methylthio group of prometryn was extended to introduce the carboxyl group to prepare prometryn hapten.The hydroxyl group of alpha-（2,4-dichlorophenyl）-1H-imidazole-1-ethanol was substituted with halogenated ester and then through the ester hydrolysis reaction to introduce the carboxyl group to prepare imazalil hapten.The carboxyl group was introduced into the benzene ring of pyrimethanil and used as the hapten.Dapsone contained the active amino group and was directly used as the hapten.The nucleophilic substitution reaction with succinic anhydride introduced hyoscyamine into the carboxyl group and was used as the hapten.The antigens were prepared by coupling the active group of haptens with carrier proteins to expose the specific antigenic epitope fully.Based on the prepared antigens of the six drugs,experiments such as animal immunization and screening,hybridoma preparation and purification were carried out,respectively.Six hybridoma cell lines were established,and six monoclonal antibodies were further prepared,namely:diamide 4F5,prometryn 1A2,imazalil 3G5,pyrimethanil 4A11,dapsone 2H9 and hyoscyamine 2F6.The types of the six antibodies belonged to Ig G1,Ig G2a,Ig G2a,Ig G1,Ig G2b and Ig G2a,respectively.The affinity constants of the antibodies were3.87×10⁹ L/mo L,3.47×10⁹ L/mo L,1.66×10⁹ L/mo L,7.03×10⁹ L/mo L,3.55×10⁹ L/mo L,2.45×10⁹ L/mo L,respectively.The six antibodies were all high-affinity antibodies.Based on the above six antibodies,enzyme-linked immunosorbent assays（ELISA）were established to detect diamide,prometryn,imazalil,pyrimethanil,dapsone and hyoscyamine after optimizing the p H value,ionic strength（Na Cl content）and organic solvent（methanol content）of the detection solution.Diamide 4F5 was a group-selective antibody that could specifically recognize four diamide drugs.The half inhibitory concentration(IC₅₀)values of cyclaniliprole,cyantraniliprole,chlorantraniliprole and tetrachlorantraniliprole were 0.16ng/m L,0.09 ng/m L,0.06 ng/m L and 0.19 ng/m L,respectively.And the linear detection ranges were 0.04～0.57 ng/m L,0.02～0.54 ng/m L,0.01～0.28 ng/m L and 0.06～0.68 ng/m L,respectively.Prometryn 1A2,imazalil 3G5,pyrimethanil 4A11,dapsone 2H9 and hyoscyamine 2F6 were monospecific antibodies with little or no cross-reactivity to other structural analogs.The IC₅₀values of prometryn,imazalil,pyrimethanil,dapsone and hyoscyamine by ELISA were 0.65ng/m L,0.95 ng/m L,0.81 ng/m L,0.59 ng/m L,0.92 ng/m L,respectively,and the linear detection ranges were 0.17～2.41 ng/m L,0.27～3.36 ng/m L,0.17～3.78 ng/m L,0.18～1.90 ng/m L and0.22～3.92 ng/m L,respectively.Fruit and vegetable samples were spiked with four kinds of pesticides and then analyzed;the recovery rates of diamide,prometryn,imazalil and pyrimethanil were 83.2%～107.7%,85.7%～108.4%,85.3%～106.7%and 84.0%～105.9%,respectively.Milk samples were spiked with two veterinary drugs and then analyzed;the recovery rates of dapsone and hyoscyamine were 89.0%～106.2%and 85.5%～93.8%,respectively.These all could satisfy the determination of drugs residues in actual samples.The six monoclonal antibodies were labelled with 18 nm colloidal gold that was prepared by the trisodium citrate reduction method.Immunochromatographic test strips were further prepared after optimizing the concentration of gold-labelled antibodies and the concentration of sprayed membrane antigens.And the immunochromatographic rapid detection methods for diamide,prometryn,imazalil,pyrimethanil,dapsone and hyoscyamine were established.The cut-off values of the test strips for detecting cyclaniliprole,cyantraniliprole,chlorantraniliprole and tetrachlorantraniliprole in fruits and vegetables were 10 ng/g,5 ng/g,2.5 ng/g and 10 ng/g,respectively.The cut-off values of the test strips were 10 ng/g for the detection of prometryn,imazalil and pyrimethanil in fruits and vegetables.The cut-off values of the test strips were 20ng/g for detecting dapsone and hyoscyamine in milk.These could all meet the market testing requirements.In conclusion,the established immunochromatographic assays could be used for high-throughput and rapid detection of diamide,prometryn,imazalil,pyrimethanil,dapsone and hyoscyamine drugs residues in food.