Rapid Immunoassay For Detecting Antiparasitic And Antipsychotic Drugs In Animal-derived Food

China is a large country of animal husbandry and consumption of animal-derived food.The abuse or illegal use of veterinary drugs is serious.Problems of food safety occur from time to time caused by excessive drug residues,which not only affects the economic development of animal husbandry,but also brings potential risks to the health of consumers.Therefore,the“Maximum residue limits for veterinary drugs in foods”in the National food safety standard was issued by the Ministry of Agriculture in 2019 and specified the residue limits and the usage of commonly used veterinary drugs.The key to monitor veterinary drugs for preventing drug abuse by relevant departments depends on efficient and practical detection technology.Immunoassay technology has the advantages of rapid analysis,simple operation,high sensitivity and low cost.Especially,the immunochromatographic strip serves as an important role in the initial screening of large quantities of samples.In this study,diminazene（DIM）,Imidocarb（IM）,halofuginone（HA）,narasin（NA）,clorsulon（CLOR）,and antipsychotic drugs of azaperone（AZN）,xylazine（XYL）and chlorpromazine（CPZ）were taken as research objects.And high affinity monoclonal antibodies against target analytes were prepared by designing and synthesizing artificial antigens.Based on these monoclonal antibodies,the corresponding rapid immunoassay method was established.（1）According to the molecular structure characteristics of different drugs,the haptens were designed and modified,and the appropriate coupling methods were selected to synthesize immunogenic artificial antigens with carrier proteins.The synthetic antigens were identified by ultraviolet-visible spectrophotometry for preliminarily identifying.（2）Based on the synthetic artificial antigens,monoclonal antibodies against DIM,IM,HA,NA,CLOR,AZN,XYL and CPZ were obtained through animal immunization,cell fusion,hybridoma cell screening,ascites preparation,and the performance of the antibodies were identified.The IC₅₀ of these antibodies were 0.37,0.074,0.49,1.70,6.46,0.35,0.15 and 0.84ng/mL,respectively;the affinity constants were 2.21×10⁸,4.58×10¹⁰,2.41×10⁸,1.31×10⁸,4.73×10⁷,1.28×10⁸,6.94×10⁷ and 1.21×10⁸L/mol,respectively;the subtypes were IgG1,IgG1,IgG2b,IgG2b,IgG1,IgG1,IgG1 and IgG2b;in specificity determination,the CR value of NA antibody to SAL was 114.86%,and the CR value of AZN antibody to AZL was 112.9%,in addition,the remaining antibodies had no cross reaction with other related compounds.（3）Based on the monoclonal antibodies of five antiparasitic drugs,the corresponding rapid immunoassay methods were developed.After optimization,the cutoff values of DIM and IM colloidal gold strips in milk were 50 ng/mL and 5 ng/mL,IC₅₀ values were 5.2 ng/mL and 0.4ng/mL,the detection limit were 1.2 ng/mL and 0.078 ng/mL,respectively;the cutoff values of HA and CLOR colloidal gold strips in bovine liver and kidney were 20 ng/g,20 ng/g,100 ng/g and 100 ng/g,IC₅₀ values were 2.46 ng/g,2.56 ng/g,14.41 ng/g and 11.45 ng/g,the detection limit were 0.44 ng/g,0.46 ng/g,2.98 ng/g and 1.85 ng/g,respectively;in ELISA method of NA,the IC₅₀ values of NA and SAL in chicken liver were 0.87 ng/g and 0.85 ng/g,detection limit were 0.11 ng/g and 0.10 ng/g,respectively.The established rapid immunoassay method could be applied to the measurement in actual samples.（4）The rapid immunoassay for AZN,XYL and CPZ were established by using three antipsychotics.The optimized results showed that the cutoff values of AZN colloidal gold strip for AZN and AZL in pork samples were both 8 ng/g,IC₅₀values were 0.74 ng/g and 0.60 ng/g,the detection limits were 0.14 ng/g and 0.12 ng/g,respectively;the cutoff values of XYL colloidal gold strip in milk samples was 5 ng/mL,IC₅₀values was 2.46 ng/mL,the detection limit was 0.44 ng/mL;the IC50 values of CPZ ELISA method for CPZ and 7-CPA in pork samples were 0.56 ng/g and 0.55 ng/g,the detection limits were both 0.05 ng/g.In conclusion,this study synthesizes small molecule artificial antigen synthesis of eight veterinary drugs,and established the corresponding rapid immunoassay method,which provided an effective tool for rapid detection of veterinary drug residues in actual samples.