Study On The Rapid Detection Technology Of The Metabolite AHD Of Nitrofurantoin

In recent years, more and more people pay attention to the veterinary drug residues infood-producing animal. It was found that large dosage or long time administration of nitrofurans toanimals will lead to a series of serious effects such as carcinogenic, meratogenic and mutageniccharacteristics. The metabolite of nitrofurans can bind to tissue proteins and may persist forconsiderable periods of time in animal tissues. As a consequence, nitrofurans have been banned forusing in animal husbandry in many countries. But due to their broad antimicrobial spectrum and lowcost, the illegal use of nitrofurans still exists in many countries. As a member of nitrofurans the toxicityis more serious than the other nitrofuran drugs except nitrofurazone.However there are nearly no reportsabout ELISA or CLIA to detect the residual of nitrofurantoin.The establishment of a rapid and sensitivedetection method for screening AHD residuals in animal products is urgently needed.In our experiments,we planned to prepare coating antigen by conjugating Jeffamine method to screen the high quality ofnitrofurantoin monoclonal antibody and establish two properly ELISA methods based on themonoclonal antibody. It was proved that the direct competitive ELISA method is better than the indirectcompetitive ELISA method via the comparison of major indexes. On the basis of the technical route ofthe former method, CLIA analysis method was also established, which provided a basis for thedevelopment of nitrofurantoin CLIA detection kit. The experiments are as follows:1. In this study, CPAHD was conjugated with protein BSA and Jeffamine-OVA byN-hyduoxysuccinimide ester method, respectively. Then the compounds were identified by UV spectrascanning methods and SDS-PAGE and the resμLts indicated that CPAHD was conjugated with BSA andJeffamine-OVA successfully. The spleen cells and the myeloma cells were fused by hybridomatechnology. Three hybridomas which produced antibody steadily from the well of1B10,3C3and3G10were characterized by indirect ELISA method. And the specificity and sensitivity of the antibodiesproduced by the three hybridomas were also characterized by indirect ELISA method. The specificityand the sensitivity of the antibodies produced by the three hybridomas were characterized by indirectELISA method. The cross-reactivity studies showed that there was no cross reactivity （CR <0.01%） ofthe monoclonal antibody with other testing compounds except CPAHD and nitrofurantoin. The halfinhibitory concentration (IC₅₀) of1B10antibody was3.56ng/mL. The results proved that the chemicalstructure of CPAHD was protected using the Jeffamine as a spacer arm between CPAHD and OVA.And CPAHD-Jeffamine-OVA as the coating antigen can be successully screened monoclonal antibody.2. The direct competitive ELISA was established by enzyme-labeled drug and antigencompetitively binding with coating antibodies in microtiter plate. The standard curve regressionequation was y=-0.3615x+0.6263（R²=0.9910）and the IC₅₀was2.236ng/mL. The indirectcompetitive ELISA was established by CPAHD-Jeffamine-OVA as coating antigen. The standard curveregression equation was y=-0.4197x+0.7343（R²=0.9985） and IC₅₀was3.616ng/mL. Through comparing the sensitivity, accuracy and stability of the two detection methods, the direct competitiveELISA method is better than other detection methods, then established the CLIA method wasestablished based on this technology.3. The CLIA method was established based on the direct competitive ELISA technology and thestandard curve regression equation was y=-0.3659x+0.5115（R²=0.9852） and IC₅₀was1.076ng/mL,the sensitivity of the method was0.303ng/mL and the detection limit of the extracted sample was0.587ng/mL.The mean recovery of AHD fortified with fish tissue was between75%-93%. Comparedwith the commercial kit, the established CLIA method with the similar sensitivity, precision andaccuracy can meet the need of detecting nitrofurantoin residues actually.