Rapid Immunoassays For Five Chemical Hazards In Food

With the increasing improvement of daily living standards,people are focusing more on the safety of food.Due to the increasingly serious phenomenon of detection of chemical hazards in food,the health of consumers has been greatly threatened.However,current instrumental analysis methods are difficult to meet the needs of on-site high-throughput detection.Therefore,the establishment of an efficient and rapid monitoring means has important practical significance and market application value.In this study,five chemical hazards in food were studied as the subjects,including four drugs illegally added in health foods（furosemide,torasemide,fenfluramine,barbiturates）and a plant growth regulator（abscisic acid）irregularly used in food.Five haptens were designed according to the above compounds.Monoclonal antibodies with high specificity and sensitivity against these five compounds were prepared,and on this basis,enzyme-linked immunosorbent assay（ELISA）and colloidal gold immunochromatographic assay were established,which were successfully applied to the analysis and detection of five chemical hazards in food.The hapten was designed and synthesized based on the chemical structure characteristics of the research object.Torasemide-hapten was prepared by adequate exposure of sulfonylureas and introducing a carboxyl group on a benzene ring;fenfluramine-hapten was designed and prepared by exposure with trifluoromethyl group in the structure as the epitope and then introducing a carboxyl group at an imino group far from the epitope;barbiturates-hapten retained the common structure of malonylurea of barbiturates to the largest extend,used phenobarbital as the core target structure,extended the carbon chain and introduced a carboxyl group as active groups and then synthesized.Furosemide and abscisic acid contained an active group carboxyl group and can be used directly as haptens.Then the active group carboxyl group was activated by carbodiimide method,and the complete antigens were prepared by conjugation with the carrier protein.Based on the successfully prepared complete antigens,five hybridoma cell lines were obtained by animal immunization,serum screening and hybridoma cell fusion,and five monoclonal antibodies were obtained by ascites preparation and antibody purification,namely:furosemide 1B1,torasemide 2H1,fenfluramine 4B10,phenobarbital 4F6,abscisic acid2G1.The isotype of antibodies against them belonged to IgG2b,IgG2a,IgG2a,IgG2b,IgG2b,and the antibodies affinity constant K_D were 4.58×10¹¹,9.36×10¹⁰,4.07×110¹⁰,4.47×10¹⁰,4.29×10¹⁰ L/mol,all of which were high affinity antibodies.Under the optimized conditions,the ELISA methods for the detection of furosemide,torasemide,fenfluramine,barbiturates and abscisic acid were established based on the above five antibodies.Furosemide 1B1,torasemide 2H1,fenfluramine 4B10 and abscisic acid 2G1were specific antibodies and the cross-reaction experiment showed that all antibodies cross-reacted with structural analogues by<1%.The fifty percent inhibition concentration(IC₅₀)of furosemide was 0.26 ng/m L,with the linear range to be 0.06～1.19 ng/m L;the IC₅₀of torasemide was 0.80 ng/m L,with the linear range to be 0.19～3.35 ng/m L;the IC50 of fenfluramine was0.34 ng/m L,with the linear range to be 0.12～0.97 ng/m L,the IC₅₀of abscisic acid was 0.65ng/m L,with the linear range to be 0.25～1.68 ng/m L.Phenobarbital 4F6 was a group-selected antibody,the IC₅₀ of phenobarbital was 17.37 ng/m L,with the linear range to be 4.63～65.15ng/m L;the IC₅₀ of pentobarbital was 23.84 ng/m L,with the linear range to be 6.17～92.15ng/m L;the IC₅₀ of amobarbital was 18.76 ng/m L,with the linear range to be 6.83～51.51 ng/m L;the IC₅₀ of p-secobarbital was 21.71 ng/m L,with the linear range to be 5.62～83.84 ng/m L.By determination of addition recovery and coefficient of variation,the accuracy of the established ELISA method was evaluated.The samples were extracted from methanol and blown dried to be diluted 10-fold with the optimized ELISA buffer solution.In the health food tablets and capsules,the recovery rate of furosemide was 94.0%～101.4%,torasemide was 98.9%～108.0%,fenfluramine was 96.0%～101.0%,barbiturates was 90.0%～107.4%,respectively;in the fruit and vegetable samples,the recovery rate of abscisic acid was 89.0%～106.2%.The assay results were consistent with LC-MS/MS results and the established method had good accuracy and can be applied to the analysis and determination of five chemical hazards in actual samples.Using the prepared five monoclonal antibodies as raw materials,the concentrations of antigens and antibodies were optimized,and colloidal gold immunochromatographic strips were developed to establish the rapid immunochromatographic detection methods for the determination of furosemide,torasemide,fenfluramine,barbiturates and abscisic acid were established in actual samples.Furosemide,torasemide,fenfluramine,barbiturates were detected in health food samples,and abscisic acid was determined in fruit and vegetable samples by immunochromatographic strips.Health food and vegetable samples were extracted by 0.01 M PBS（containing 20%methanol）and diluted twice with sample diluent（0.01 M PBS,p H 7.4,containing 1%ON-870）to detect,and fruit samples were extracted by 0.01 M PBS（containing20%methanol）,adjusted to neutral p H and diluted 3 times to detect.The results showed that the cut-off values of furosemide,torasemide and fenfluramine in health food samples were 10ng/g,phenobarbital was 100 ng/g,pentobarbital and amobarbital was 500 ng/g,secobarbital was 200 ng/g,abscisic acid was 10 ng/g,which could meet the detection requirements.The results showed that the established method of immunochromatographic strips can be applied in the high-throughput and rapid screening of furosemide,torasemide,fenfluramine,barbiturates and abscisic acid in food.