Rapid Immunoassay For 13 Typical Chemical Residues In Animal-derived Food

This paper focuses on 13 chemical residues that are easily abused or illegally added in animal-derived foods.Monoclonal antibodies were prepared and a sensitive and rapid detection technique was established and applied to the detection of actual animal-derived samples.The main research content and results are as follows:1.Analysis of the structural characteristics of chemical residues,design and preparation of hapten.（1）12 specific haptens were developed for 10 chemical residues,including nicarbazin marker,virginiamycin M1,quintozine,roxarsone,moroxydine,beta（2）agonists,stanozolol,estradiol benzoate and testosterone propionate by modification of their parent nucleus structures,including reduction of nitro to amino,condensation of hydroxyl groups with anhydride to introduce carboxyl groups and condensation of carbonyl groups with hydroxylamine.（2）The structural characteristics of the hapten of Imidocarb,diclofenac,and Zilpaterol were analysed and the drug intermediates were modified by introducing reactive groups to develop 3 specific haptens.The 15 haptens developed were characterised by various characterisation tools such as LC-MS/MS and 1H NMR,indicating the haptens were successfully derived.2.Artificial complete antigen preparation and animal immunisation.Depending on the active groups（-COOH,-NH₂,-OH）after derivatization,coupling methods such as activated ester,carbodiimide,mixed anhydride,diazo and other coupling methods were used to couple with carrier proteins BSA,KLH and OVA and characterized by UV spectrophotometry and SDS-PAGE gel electrophoresis.The antibody titres of the sera were determined by multiple immunization of BALB/c mice,and mice with high potency and good inhibition of the target molecules were screened for cell fusion,and monoclonal antibodies were screened by hybridoma cell technology.3.Preparation and characterisation of high affinity monoclonal antibodies.The hybridoma cells were domesticated using chicken and pork extracts to screen for hybridoma cell lines that secrete high-affinity antibodies.13 high-affinity monoclonal antibodies were obtained after three clonal screens,cell line expansion,ascites preparation and purification of:3A3（salinomycin and narasin）,3G3（nicarbazin marker）,4H12（virginiamycin M1）、2E9（imidocarb）、2H11（quintozine）、3F2（roxarsone）、3B6（diclofenac）、4A7（moroxydine）、2G5（zilpaterol）、3F10（beta（2）agonists）、3E6（stanozolol）、2H2（estradiol benzoate）and 4E1（testosterone propionate）.The half inhibition rates(IC₅₀)of the 13 monoclonal antibodies mentioned above ranged from 0.17 to 5.86ng/m L,and the affinity constants of the antibodies all ranged from 10⁹to 10¹⁰L/mol.3F2,3B6,4E1 and 3F10 are broad-spectrum monoclonal antibodies,of which ROX-m Ab 3F2recognises 4 residues including roxarsone;DIC-m Ab 3B6 recognises 3 residues including diclofenac;Tp-m Ab 4E1 recognises 4 residues including testosterone propionate;andβ（2）agonists-m Ab 3F10 recognises 12 beta（2）agonists including salbutamol.4.Development of immunochromatographic strip assays in animal-derived food.By optimizing the p H of the colloidal gold-labeled antibody,coating antigen concentration,amount of gold-labeled antibody added,and suspension of the gold-labeled antibody,colloidal gold immunochromatography test strips（ICS）for 13 chemical residues detection were developed and applied to detect chicken,pork,beef,lamb,and swine urine.The visual limit of detection（v LOD）for chicken ranged from 0.25～2.5μg/kg,with a maximum quantitative limit（LOQ）of no more than 1.76μg/kg;the v LOD for pork was 1～25μg/kg,with a maximum LOQ of 1.50μg/kg;the v LOD for beef was 1～5μg/kg,with a maximum LOQ of 0.36μg/kg;the v LOD for lamb was 0.25～2.5μg/kg,with the LOQ value of 0.12μg/kg;the v LOD for swine urine was 0.1～2.5 ng/m L,with an LOQ value of 0.08ng/m L.All values met the national food safety limit standards.Testosterone propionate,stanozolol and estradiol benzoate multiplex immunochromatographic test strips（Multi-ICS）were developed by setting up multiple chromatographic lines of detection.ICS and LC-MS/MS tests were performed on spiked and market collected samples and the immunochromatographic strip method established was in perfect agreement with the instrumental assay.In summary,the specific monoclonal antibodies were developed in this dissertation,immunochromatographic technique for 13 chemical residues commonly found in animal-derived foods.The assay is suitable for on-site screening and has the advantages of high sensitivity and stability,providing a powerful tool for national prevention and control of animal-derived food safety.