Development Of Fluorescence And Chemiluminescence Immunoassays For The Determination Of Pesticide Residue

On the basis of the existing research of the author's laboratory about the ELISA method,in-depth study about the effect of various factors which including matrix effect,drift phenomenon and different ELISA coating formats to ELISA method was investigated.Comparison of different ELISA formats showed that antibody coated format and the second antibody coated format had better performance in sensitivity and variation coefficient of determination than antigen coated format.In order to establish the immuno-chemical method which had higher sensitivity and good application prospect,the systematic experiments about time-resolved fluorescence immunoassay(TRFIA) and chemiluminescence ELISA immunoassay(CLEIA) were carried out.A variety of conditions of fluorescence enhancement solution including the concentration ofβ-NTA,concentration of TOPO and pH value were optimized, subsequently,the fluorescence enhancement solution which had good enhancement activity to both Eu³⁺ and Sm³⁺ was obtained.Based on the results,TRFIA conditions were optimized.On the basis of the optimized conditions mentioned above,the carbofuran TRFIA method was generated and recoveries of standards was determined. The sensitivity for detection,the IC₅₀ value was 38.55 ng/ml at a practical working concentration range from 1.86 to 798.8 ng/ml,and the limit of detection for carbofuran was 1.86 ng/ml.The mean variation coefficient of standard determination was 25.30%.Continued,analysis of carbofuran in spiked sample was finished. Unfortunately,there was no advantage about the carbofuran TRFIA method to the carbofuran ELISA method.Therefore,the research on carbofuran CLEIA method was carried out.The chemiluminescence sensitizer was obtained after a variety of conditions were optimized.On the basis of it,CLEIA method conditions for earbofuran was optimized and the optimum conditions were given as followed:Costar^? white plate for chemiluminescence was coated with the antibody in 0.1M,pH7.2 Tris-HC1 and the plate was incubated at 37℃for 2 hours.Also,1%typtone as blocking substance and 0.05 M,pH7.2Tris-HCl buffer as reacting buffer could improve the method's sensitivity and get the largest light-emitting count.And 2.5%methanol was suitable for the competitive reaction.On the basis of optimum conditions,the carbofuran CLEIA method was generated successfully.The sensitivity for detection,the IC₅₀ value was 4.244 ng/ml at a practical working concentration range from 0.362 to 49.76 ng/ml and the limit of detection for carbofuran was 0.362 ng/ml.Recoveries of carbofuran spiked into vegetable samples and environmental samples were determined by CLEIA after sample matrix effect testing.Finally,the confirmation test between CLEIA and LC-MS/MS was finished.The results showed that CLEIA method had higher sensitivity than ELISA method,and the limit of detection of a variety of samples reached the carbofuran's MRL value laid down by CAC and relevant countries.On the success in generating the chemiluminescence immunoassay system,the analytical method was applied in determination of organophosphorus pesticides including triazophos,chlorpyrifos and parathion.The IC₅₀ value of CLEIA methods in determination of triazophos,chlorpyrifos and parathion were 0.852,21.01 and 2.247ng/mL,anad IC₁₀ value were 0.063,0.694 and 0.203ng/mL respectively. Subsequently,the results were compared with the results of ELISA methods.It showed that the LOD of CLEIA methods have been greatly improved,especially the triazophos CLEIA method.Compared to the ELISA method,the detection sensitivity and LOD were improved two orders of magnitude.It indicated that the CLEIA method had good prospects of application in detection of pesticide residue.