Establishment And Evaluation Of Rapid Quantitative Detection Of Chlorpromazine In Animal-derived Foods By Time-resolved Fluorescence Immunoassay

Chlorpromazine(CPZ)belongs to the class of phenothiazines and is mainly used in the treatment of mental illness.It is also used in veterinary clinics due to its strong sedative and antiemetic effects.At the same time,the addition of CPZ in the feed can play the role of sedative hypnosis,weight gain and fattening,and shortening the time of release.However,if CPZ remains in animal products,it will have adverse reactions to consumers and affect human health.Although China has banned the use of CPZ for food animals,there are still illegal traders who illegally add CPZ to food animal feed for the benefit of food.In order to better protect people’s health and promote the import and export of animal-derived foods,it is urgent to establish a simple,rapid and effective method for detecting CPZ residues.Therefore,this paper applies the advantages of sensitive,specific,rapid and simple immunoassay technology to the field of drug residue detection,and initially establishes time-resolved fluoroimmunoassay(TRFIA)for rapid quantitative detection of CPZ.The accuracy of the method was evaluated by actual sample detection and comparison with ultra performance liquid chromatography-tandem mass spectrometry(UTM-MS/MS).The results show that the established method has high accuracy,simple operation and large detection flux,which is suitable for rapid screening of CPZ.First,the complete antigen of CPZ-bovine serum albumin(BSA)was synthesized by carbodiimide method(EDC).The complete antigens of CPZ,BSA and CPZ-BSA were scanned in the ultraviolet-visible light at 200～400 nm.The UV spectra of the three antigens were analyzed.The complete antigen-conjugate of CPZ-BSA also possesses the characteristic absorption peaks of CPZ and BSA,indicating the CPZ whole antigen.The preliminary synthesis was successful.Balb/c mice were immunized with CPZ-ovalbumin(OVA)complete antigen to prepare anti-CPZ monoclonal antibodies.The specificity of the CPZ monoclonal antibody was evaluated by electrophoresis and by enzyme linked immunosorbent assay(ELISA).The results showed that the CPZ monoclonal antibody was of high purity and specificity,and was suitable for the establishment of immunological methods.On the basis of synthesizing CPZ complete antigen and preparing monoclonal antibody,a method for rapid quantitative detection of CPZ by TRFIA was established.The CPZ monoclonal antibody was coupled with Eu-fluorescent microspheres to prepare a fluorescent microsphere probe;CPZ-BSA and goat anti-mouse secondary antibody were sprayed on a nitrocellulose filter membrane(NC membrane),respectively.Detection line(T line)and quality control line(C line).The particle size of the fluorescent microspheres was selected,and the conditions of fluorescence microsphere dilution,fluorescent microsphere antibody labeling,NC membrane chromatography speed and NC membrane T-line CPZ-BSA total antigen concentration were optimized.The optimal conditions were as follows:Select 300 nm fluorescent microspheres with a dilution factor of 1:800,an antibody labeling amount of 30 μg·mL-1,a chromatographic speed of the NC membrane of 135 s-4 cm-1,and a T-line total antigen concentration of 1.0 mg·mL-1.Under the optimal conditions,the CPZ quantitative standard curve was established with a linear range of 0.0625-10 ng·mL-1.The detection limit was 0.35 ng·mL-1,the coefficient of variance(CV)was 6.32%,the coefficient of variation between batches was 8.36%,and the standard addition was between 92.7 and 101.6%.In the stability test,the CV did not change significantly after half a year of storage at room temperature.Finally,UPLC-MS/MS method was used to confirm the established TRFIA quantitative detection CPZ method.The comparison of the spiked recovery rate proved that the established TRFIA method has good accuracy.The method of CPZ fluorescence immuno-immunoassay established in this paper is rapid,sensitive,wide linear range and specific.It can be used for the rapid detection of CPZ residues in pork.It can also be used as an effective supplement to the instrumental analysis method,and also provides a powerful reference for CPZ detection in different animal source foods in the future.