Immunoassay Methods Development For The Determination Of Cyproheptadine

In recent years,food safety issue has been concerned publicly,animal drug illegal using or abusing would cause serious problems for human health.Since clenbuterol has been banned for animal using,there is a new situation emerging with cyproheptadine(CYP)illegally used as animal drug.As a prescription drug,cyproheptadine is registered only as an anti-allergic treatment for human deseases,however,if illegally added in animal feed,it also take similar effect to animals as clenbuterol does,thereby,risking the animal product safety.Kids and old people are the most of sensitive population,high concentration of cyproheptadine could cause coma,shortness of breath,even death.Thereby,cyproheptadine is prohibited from being used as a veterinary drug world wildly.The Ministry of Agriculture of People's Republic of China also issued the Announcement No.1519 on December 27,2010,prohibiting the use of cyproheptadine in animal feed and drinking water.Antibody based Immunoassay method has been wildly used for the drug residue detection,which is sensitive,specific,rapid and simple.So far,there is no antibody based immunoassay has been reported for cyproheptadine detection,it is urgent to provide a simple and convenient detection method for cyproheptadine detection.In this study,a derivative of cyproheptadine has been structurally modified and synthesized,and conjugated to carrier protein using as immunogen.Thereafter,polyclonal and monoclonal antibody has been produced and characterized for the immunoassay method development.The main results are as follows:Part ?: Hapten synthesis and carrier protein conjugation:2-methyl-4-(5H-dibenzo [a,d] cycloheptatrien-5-ylidene)piperidineacetic acid was successfully synthesized,and structurally confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry.Two types of complete antigens CYP-KLH?CYP-BSA and coating antigen CYP-OVA were successfully achieved by conjugating cyproheptadine with three carrier proteins KLH,BSA and OVA,respectively.Part ?:Polyclonal antibody production and in-direct enzyme linked immunosorbent assay(id-ELISA)development:for antigen CYP-BSA immunization,Polyclonal antibody obtained with the titer above 10000,No.1 mouse was 64000-128000 X,No.2 mouse was 128000 X,No.3 mouse was 32000-64000 X.The ELISA results showed that half-inhibitory concentration(IC50)of cyproheptadine was 0.02?g/mL for No.1 mouse,93.95?g/mL for No.2 mouse,2.53?g/mL for No.3 mouse.IC50 of cyproheptadine hapten was 0.032?g/mL for No.1 mouse,1.61?g/mL for No.2 mouse,0.492?g/mL for No.3 mouse;antiserum titers of CYP-KLH immunization reached to 16000 X for No.1 mouse,8000-16000 X for No.2 mouse,8000-16000 X for No.3 mouse,respectively.The IC50 of cyproheptadine was 2.685?g/mL for No.1 mouse,2.328?g/mL for No.2 mouse,2.999?g/mL for No.3 mouse.For cyproheptadine hapten,IC50 was 0.242?g/mL for No.1 mouse,0.261?g/mL for No.2 mouse,and 0.132?g/mL for No.3 mouse.Part ? : Monoclonal antibody production and characterization: Hybridoma cells were prepared by mixing mouse spleen cells with myeloma cells SP2/0.Through limiting dilution analysis and subcloning,unfortunately,none of stable cell line has been obtained at this moment.