Deltamethrin Enzyme-linked Immunosorbent And Colloidal Gold Labeling Immunoassay

The over use of deltamethrin made the great issue of residue exceeded.The anti?Del antigen and enzyme labeled Del hapten from our laboratory were employed to establish for rapid detection of deltamethrin residue immunoassay.After the conditions optimization,the antibody solution of 2?g/mL was used to coat ELISA plate and the Del-HP-HRP was diluted 2.0×105 times in dc-ELISA.The proper medium for antibody immobilization was pH7.2 phosphate buffer(PB),and the pH6.8 PB with 0.125mol/L NaCl was used as reaction medium.Under the optimized ELISA conditions,the Del inhibition standard dc-ELISA curve was established.The results showed that the linear concentrations for Del detection ranged from 10?g/mL to 0.10?g/mL,and the regression equation of Del inhibition curve was I=0.3930p+0.4114,the square of correlation coefficient(R2)was 0.9683,the half-maximal inhibition concentration(IC50)was 1.68?g/mL with relative standard deviation(RSD)of 2.11%(n=5),and the limit of detection(IC10)was 0.161 ?g/mL.The blank extract of pakchoi cabbage was used as reaction medium to established Del dc-ELISA working curve,the results showed that the linear concentrations for Del detection was ranged from 30?g/mL to 0.1?g/mL,and the regression equation of Del working curve was 1=0.324p+0.3651?the square of correlation coefficient(R2)was 0.9803,the half-maximal inhibition(IC5,)was 2.60?g/mL with the RSD of 13.7%,and the limit of detection(IC10)was 0.152?g/mL.The Del standard was added into pakchoi cabbage at the levels of 5mg/kg,1mg/kg and 0.2mg/kg respectively.The recoveries determined by the method of dc-ELISA were 97.0%with the RSD of 8.5%(n=5)at the spiked level of 5mg/kg,106%with the RSD of 8.1%(n=5)at the spiked level of 1mg/kg,and 95.4%with the RSD of 8.1%(n=5)at the spiked level of 0.2mg/kg.The conditions of Del ic-ELISA were optimazed by the way of phalanx test,the concentrations of coating antigen and antibody were both 0.1?g/mL.Under the optimized ELISA conditions,the Del inhibition standard ic-ELISA curve was established.,the linear concentration of Del ranged from 10?g/mL to O.10?g/mL.The regression equation was 1=00.4365p+0.3848,the relative coefficient(R2)was 0.9794,the half-maximal inhibition concentration(IC50)was 1.84?g/mL with the RSD of 13.8%,and the limit of detection(IC10)was 0.223?g/mL.The blank abstract of pakchoi cabbage was used as reaction medium to establish Del ic-ELISA working curve,the regression equation was 1=0.4322p+0.5363,the relative coefficient(R2)was 0.9936,and the linear concentrations for Del detection ranged from 10?g/mL to 0.1?g/mL,the half-maximal inhibition concentration(IC50)was 0.824?g/mL with the RSD of 9.54%and the limit of detection(ICio)was 0.098?g/mL.The Del standard was added into pakchoi cabbage at the levels of 5 mg/kg,1mg/kg and 0.2mg/kg respectively,the recoveries were 82.1%with the RSD of 2.21%(n=5)at the spiked level of 5mg/kg,87.3%with the RSD of 6.57%(n=5)at the spiked level of 1mg/kg,and 106%with the RSD of 10.6%(n=5)at the spiked level of 0.2mg/kg.The colloidal gold was prepared by reducing the chloroauric acid with different amount sodium citrate,the result showed that:3ml 1%sodium citrate in 100ml 1%chloroauric acid is the proper reaction concentration.The optimum pH for anti-Del antibody labeling was 7.5,and the optimum amount of anti Del antibody was 12?g/mL.The proper sample pad and gold conjugate pad were both the KB50 glass fiber,and the CN140 nitrocellulose membrane was chosen as chromatography membrane.The antigen of 100?g/mL and sheep anti-rabbit IgG of 500?g/mL was used to line onto the NC membrane with the amount of 1.25 ?L/cm and an interval of 8mm to serve as test line and control line respectively.The 0.Olmol/L pH7.5 PB was used as chromatographic medium,proper time for the immunochromatography was 10-15min,the sensitivity of colloidal gold abeled immunochromatography for Del detection was 0.1?g/mL,it is suitable for the rapid detection of Del residue.The Del direct competitive enzyme-linked immunosorbent assays(dc-ELISA)?indirect competitive enzyme-linked immunosorbent assays(ic-ELISA)and colloidal gold immunochromatography assay(GICA)were successfully developed,among them,the dc-ELISA detection and GICA specific for deltamethrin has not been reported.