Rapid Immunoassay For Five Chemical Drugs Residues In Animal-Derived Food

In the process of animal breeding,the abuse or illegal use of antibiotics and coccidiostats is one of important factors for causing animal food safety problems.In this study,antibiotics?Lincomycins,Macrolides,Carbadox and Cyadox?and coccidiostats?Madumycin and Benzimidazoles?in common use were chosen as research objects,with the purpose of establishing colloidal gold immunochromatographic assay?GICA?by hapten design and specific or group selective monoclonal antibody preparation.Three kinds of lincomycins haptens and their corresponding complete antigens were designed and synthesized.Through mouse immunization,serum screening,cell fusion and ascites preparation,the monoclonal antibody that can simultaneously recognize clindamycin?CLIN?,lincomycin?LIN?and pirlimycin?PIR?were obtained.With the optimization of standard dilution buffer,half inhibitory concentration(IC₅₀)of this antibody for CLIN,LIN and PIR were 0.42,3.27 and12.8 ng/mL,respectively.The affinity constant?Ka?was 1.36×10¹¹ L/mol.Subtype was Ig2a.It had no cross-activity with other antibiotics.Based on this antibody,the GICA was eastablished for CLIN,LIN and PIR residues in milk,egg,beef and honey.Using the strip scanner,the calculated limit of detection?cLOD?for CLIN,LIN and PIR were 0.04,0.43 and 7.18?g/L in milk;0.81,1.36 and 11.06?g/kg in egg;1.54,1.43 and10.58?g/kg in beef;0.16,3.65 and 20.91?g/kg in honey.These meet the requirement of the maximum residue limits?MRLs?for lincomycins in food.Confirmed by positive honey sample,this developed GICA could be used for CLIN,LIN and PIR residues detection in food.For spiramycin?SP?,kitamycin?KIT?and clarithromycin?CLA?,the haptens of these three macrolides were synthesized using oximation reaction.Then the SP?KIT and JOS antibodies were prepared,with IC₅₀ of 0.43?1.49 and 0.16 ng/mL.The SP anibody was highly specific and had no cross-activity with other macrolides,Ka was 4.52×10¹⁰ L/mol,and subtype was IgG2b.The KIT antibody had good cross-activity with josamycin(JOS,with IC₅₀ of 2.67 ng/mL),Ka was 1.19×10⁹ L/mol,and subtype was IgG2a.The CLA antibody had good cross-activity with erythromycin?ERY?,roxithromycin?ROX?,azithromycin?AZI?,with the IC₅₀ of 0.52,0.76,and 0.99 ng/mL.Ka was 4.09×10⁹ L/mol,and subtype was IgG1.Based on SP antibody,the GICA was eastablished for SP residues in milk and beef,with cLOD of 0.22?g/L and 0.27?g/kg.Under the concentration of 0.5,1?g/L in milk and 1,5?g/kg in beef,the spiked recovery ranged from 82.3%±15.7%to 115%±8.14%,with the variable coefficient?CV?ranging from 5.2%to 19.1%.Based on KIT antibody,the GICA was eastablished for KIT and JOS residues in milk and beef,with cLOD of 1.51 and 1.91?g/L in milk,3.00 and 2.73?g/kg in egg.Under the concentration of 4,16?g/L in milk and 12,48?g/kg in egg,the spiked recovery ranged from 79.3%±9.2%to 110%±4.7%,with the CV values ranging from 4.3%to 13.8%.Based on CLA antibody,the GICA was eastablished for CLA,ERY,ROX and JOS residues in milk,with cLOD of 0.095,0.085,0.055 and 0.17?g/L.Under the concentration of 1,2.5?g/L for CLA,1,5?g/L for ERY and ROX,and 5,10?g/L for AZI,the spiked recovery ranged from 83.0%±8.0%to 102.0%±6.0%,with the CV values ranging from 3.4%to 9.8%.All spiked samples were confirmed by LC-MS/MS method,and this developed GICA method could be used for seven macrolides residues detection.The novel hapten was desigen on account of CBX and CYA chemical structure analysis.Then the monoclonal antibody that can recognized CBX and CYA simultaneously were prepared.The IC₅₀ of this antibody for CBX and CYA are 1.84 ng/mL and 1.85 ng/mL.Ka was 3.19×10⁹ L/mol,and subtype was IgG2a.The cross-activity was 7.30%with QCA,and it had no cross-activity with other quinoxalines.Based on this antibody,the GICA was eastablished for CBX and CYA residues in chicken,with cLOD of 2.92?g/kg and 2.68?g/kg.Under the concentration of 4,8 and 12?g/kg,the spiked recovery ranged from 89.5%±8.9%to 109.6%±10.3%,with CV value ranging from 9.0%to 14.6%.Confirmed by LC-MS/MS method,this GICA could be used for CBX and CYA residues detection in chicken.The specific maduramicin?MD?antibody was prepared using MD as hapten,with IC₅₀of 3.75 ng/mL.The Ka was 3.70×10¹⁰ L/mol,and subtype was IgG3.It had no cross-activity with other polyether antibiotics.Based on MD antibody,the GICA was eastablished for MD residues in chicken and egg,with cLOD of 5.51?g/kg and 6.46?g/kg.Under the concentration of 10 and 20?g/kg,the spiked recovery ranged from 88.7%±3.9%to 105.6%±5.4%,with CV value ranging from 4.4%to 5.2%.Confirmed by LC-MS/MS method,this GICA could be used for MD residues detection in chicken and egg.The group selective benzimidazoles?BZs?monoclonal antibody was prepared using2-methoxycarbonylamino-1H-benzoimidazole-6-carboxylic acid as hapten.The IC₅₀ of this antibody for albendazole?ABZ?,albendazole sulfoxide?ABZSO?,albendazole sulfone?ABZSO₂?,fenbendazole?FBZ?,fenbendazole sulfone?FBZSO₂?,flubendazole?FLU?,mebendazole?MBZ?,parbendazole?PBZ?,oxfendazole?OFZ?,oxibendazole?OXI?,carbendazol?CAR?were 0.66,2.91,5.34,0.75,6.27,0.37,0.30,1.13,19.99,0.64 and 14.84ng/mL.Ka was 1.07×10¹⁰ L/mol,and subtype was IgG2a.Based on this antibody,the cLOD of ABZ,ABZSO,ABZSO₂,FBZ,FBZSO₂,FLU,MBZ,PBZ,OFZ,OXI,CAR were 0.83,0.77,1.83,0.98,7.67,3.50,3.96,5.72,0.92,0.59,1.69?g/L,respectively.This proposed GICA can satifiy the detection requirement.