Rapid Immunoassay For Detecting Four Important Kinds Of Antibiotics

Antibiotic residues in milk has been a major threat to the safety of dairy products.Traditional assays depended on instruments are sensitive and precise.However,as the force of supervision increasing,the market and related department need more sensitive,more fast and high throughput detection methods to monitor antibiotic residues in milk.In this study,four important antibiotics residues including sulfonamides,tetracyclines,sulfonamides synergists and natamycin were considered as research objects,aiming to develop the monoclonal antibodies?m Ab?for these drugs and establish the fast immunoassays for the food safety detection.Firstly,seven kinds of haptens and corresponding antigen for sulfonamides were designed and synthesized.Two mAb 2G3 and 3D1 with different recognition were developed.For mAb 2G3,the immunogen is S1-BSA,coating antigen is homogenous conjugate S1-OVA,the subtype of mAb is IgG1.Ic-ELISA was developed for sulfamethoxazole?SMZ?based on m Ab 2G3,IC₅₀ of m Ab 2G3 against SMZ is 0.15?g/L,working range is 0.03-0.63?g/L,coefficient of variation?CV?is 0.992.For mAb 3D1,the immunogen is S5-BSA,the coating antigen is the heterogenous conjugate S3-OVA.Ic-ELISA was developed for sulfamethazine?SM₂?,IC₅₀ of mAb 3D1 against SM₂ is 0.51?g/L,working range is 0.11-2.33?g/L,CV is0.992.The recoveries of SMZ and SM₂ in milk were respectively 89.6%-119.0%and95.3%-118.0%.Secondly,colloidal gold strip test based on mAb 3D1 was established for detection of twenty-seven sulfonamides in honey samples.The optimal conditions for nanoparticles coupled with mAb were as follow:1 mL of colloidal gold solution was added with 6?L of0.1 M K₂CO₃ and 50?L of 0.4 mg/mL mAb 3D1.The optimal coating antigen concentration was 0.2 mg/mL.The cut-off values of strip test ranged from 2.5?g/kg to 100?g/kg.The limits of detection?LOD?of the test ranged from 1?g/kg to 25?g/kg.Thirdly,three immunogens were synthesized for the immunization,aiming to prepare the broad-specific mAb towards tetracyclines.Through immunogen?TC-BSA?and homogenous coating antigen?TC-OVA?,the mAb 5B11 was screened.Based on mAb 5B11,ic-ELISA was established for tetracyclines detection in milk and honey samples.The optimal conditions of ELISA were as follow:the concentration of coating antigen and mAb were 0.25?g/mL and 0.125?g/mL respectively.The IC₅₀ of mAb towards tetracycline?TC?was 0.41?g/L,working range is 0.18-3.09?g/L.The cross-reactivity of mAb 3D1 towards oxytetracycline?OTC?,chlorotetracycline?CTC?and doxycycline?DC?was 87.2%,22.5%,18.9%.The result of recovery test for TC,OTC,CTC,DC spiked in negative milk and honey samples were as follow:for milk samples:93.5%-101.2%,95.3%-105.1%,95.6%-101.5%,89.9%-92.3%,for honey samples:95.6%-100.5%,89.5%-101.3%,91.8%-103.7%,89.7%-102.3%.Fourthly,colloidal gold strip tests were developed for TCs detection in milk and honey samples.The optimal condition of coupling as follow:1 mL of colloidal gold solution was added with 6?L 0.1 M K₂CO₃ and 50?L 0.2 mg/mL m Ab 5B11.The concentration of coating antigen was 0.4 mg/mL.The cut-off values of strip test for TC,OTC,CTC and DC in milk samples were 10?g/L,10?g/L,20?g/L,25?g/L.The LOD of test were 0.5?g/L,0.5?g/L,1?g/L and 5?g/L.The cut-off values of test for TCs in honey samples were 20?g/kg,20?g/kg,40?g/kg,40?g/kg.The LOD of test were 0.8?g/kg,2?g/kg,4?g/kg and 4?g/kg.Fifthly,antigens for trimethoprim?TMP?,diaveridine?DVD?and ormetoprim?OMP?were synthesized respectively.The corresponding specific mAb was developed for three drugs.Based on mAb 2A4 for TMP,ic-ELISA was developed.The optimal concentration of coating antigen and mAb was 0.1?g/mL and 0.1?g/mL.The optimal drug diluent was 0.01 M PBS?pH 7.2,NaCl 0.8%,acetonitrile 10%?.The IC₅₀ for TMP is 4.14?g/L,working range is0.18-3.09?g/L,CV is 0.995.The recovery for TMP spiked in milk,honey and fish samples were 103.5%-118.0%,100.3%-107.1%,91.5%-108.1%.Based on mAb 3E2 for DVD,ic-ELISA also was developed with optimal conditions:concentration of coating antigen and m Ab were 0.1?g/mL and 0.1?g/mL,drug diluent was 0.01 M PBS?pH 7.2,NaCl 0.8%?.The IC₅₀ for DVD is 0.43?g/L,working range is 0.13-1.38?g/L,CV is 0.99.The recovery for DVD spiked in milk and honey sanples was 93.9%-113.8%and 89.0%-109.8%.Based on m Ab 1G10,ic-ELISA was developed with optimal conditions:coating antigen and coating antigen were 0.3?g/mL and 0.03?g/mL,drug diluten was 0.01 M PBS?pH 7.2,NaCl 1.6%?.The IC₅₀ for OMP is 5.14?g/L,working range is 1.44-20.47?g/L,CV is 0.998.The recovery for OMP spiked in milk and honey samples was 88.9%-107.6%and 113.9%.Sixthly,strip test was established for TMP and DVD based on mAb 2A4 and 3E2.For m Ab 2A4,the optimal condition of coupling as follow:1 mL of colloidal gold solution was added with 6?L 0.1 M K₂CO₃ and 50?L 0.4 mg/m L mAb 2A4.The optimal concentration of coating antigen was 0.3 mg/mL.The cut-off values of TMP spiked in milk and honey were 25?g/L and 25?g/kg,LOD were 2.5?g/L and 2?g/kg.For mAb 3E2,the optimal condition of coupling as follow:1 m L of colloidal gold solution was added with 4?L 0.1 M K₂CO₃ and 50?L 0.2 mg/m L mAb 3E2.The optimal concentration of coating antigen was 0.2 mg/mL.The cut-off values of DVD spiked in milk and honey samples were 5?g/L and 10?g/kg,LOD were 0.5?g/L and 1?g/kg.Lastly,a specific mAb 3E5 against natamycin?Nata?was developed and ic-ELISA was established.The concentration of coating antigen and mAb 3E5 was 0.1?g/mL and0.03?g/mL.The optimal drug diluent was 0.01 M HEPES?pH 7.2,methanol 5%?.The IC₅₀for Nata was 1.69?g/L,working antigen was 0.64-4.64?g/L,CV is 0.997.The recoveries of Nata spiked in milk,juice,yoghurt and cheese were 102.5%-120.5%,102.6%-121.1%,83.9%-113.8%,88.7%-108.2%.The colloidal gold strip test also was established for Nata based on mAb 3E5.The optimal condition of coupling as follow:1 mL of colloidal gold solution was added with 4?L 0.1 M K₂CO₃ and 50?L 0.4 mg/mL mAb 3E5.The optimal concentration of coating concentration was 0.2 mg/m L.The cut-off values of Nata spiked in milk and honey samples were 20?g/L and 125?g/kg,LOD were 2?g/L and 25?g/kg.