Establishment And Evaluation Of Indirect Competitive ELISA Method For Diazepam Residue Detection

Diazepam(DZP)is a common sedative and hypnotic drug.If it is eaten by livestock,it can increase the meat yield and increase the economic profit.However,there are still aquaculture companies that use diazepam for livestock production in order to make high profits.At present,there are many methods for detecting residues in diazepam.High performance liquid chromatography,gas chromatography-mass spectrometry,liquid chromatography-mass spectrometry,and enzyme-linked immunosorbent assay(ELISA)are commonly used.These methods can be used for diazepam residue detection,but the disadvantages are long pre-treatment time,high detection cost,strict knowledge of the operator’s knowledge and professional requirements,inconvenient for large-scale detection and movement difficulties.The ELISA method is simple in operation,low in detection cost,strong in specificity and high in sensitivity,and is suitable for rapid and effective determination of large-volume samples,and is not limited to laboratory testing,and is more suitable for general promotion than the above several methods.The purpose of this experiment is to have a clear understanding of the establishment of indirect competitive ELISA methods and the process of condition optimization.Ability to conduct experiments independently and conduct practical applications,and evaluate the established methods.Ability to conduct experiments independently and conduct practical applications to evaluate established methods,to lay the foundation for the study of diazepam immunoassay and the development of ELISA kits.1.Establishment of an indirect competitive ELISA assay for diazepam.When an indirect competitive ELISA method is established,the measurement is reversed and the experimental conditions are appropriately optimized.The optimum working concentration for the determination of the enzyme-labeled secondary antibody is 1:50000,and the optimum color development time is 15 min.The optimal coating condition is overnight coating at 4℃.The optimal blocking condition is blocking at 37℃ for 90 min.Using DZP-BSA as the coating original,the best working concentration of the coating was 1:10000 using the checkerboard method,and the optimal working concentration of the antibody was 1:10000.Using the diazepam standard as the drug inhibition standard curve,the linear equation is y=A2+(A1-A2)/[1+(x/x0)p],R2=0.998,and the IC50 is 1.667 ng/m L.The detection range is 0.194 ng/m L~14.364 ng/m L.2.Evaluation of indirect competitive ELISA for diazepam monoclonal antibodies.The cross-reactivity rate of 30 benzodiazepines with DZP structure similar to DZP was detected,and finally 7 cross-reactive drugs were obtained,they are Temazepam,Nimetazepam,Clonazepam,2-Hydroxyethylflurazepam,Tetrazepam,Alprazolam.(Alprazolam),Prasepam.The cross-reaction rate of chlordiazepoxide was 1.236%,and the cross-reaction rate of the other 22 drugs was extremely low(less than 0.1%).Using the established indirect competitive ELISA method to detect the residues of diazepam in pig urine,centrifuge the fresh pig urine,discard the urine sediment and use the supernatant for testing.The above measured antigen-antibody optimal working concentration was determined.After the completion of the measurement,the recovery rate in pig urine was calculated to be 91.4%~103.70%,and the intra-assay coefficient of variation(CV)was 1.98%~5.06%.The coefficient of variation(CV)was 4.87%~8.22%,and the detection limit was 0.307 ng/m L.The conclusion is to establish an indirect competitive ELISA assay for DZP monoclonal antibody,optimize some experimental conditions,and successfully applied to the detection of diazepam residues in pig urine.