Preparation Of The Anti-Coccidiostat M Ab And Its Application In Electrochemical Immunosensor

There are two types of anticoccidial drugs which include polyether ionophore antibiotics and chemosynthesis antibiotics,both them play an important role to treat coccidiosis infections.Although anticoccidial drugs are effective in the prevention and treatment of coccidiosis,their long-term,continuous addition in feed,or irregular use,can easily cause drug residues in animal-derived food products.It takes a potential risk for food safety and public health safety,thereby threatening consumer health.Hence,many countries regulate the maximum residue limits（MRLs）.Some desirable detection methods are needed to be designed for monitoring anticoccidial drugs.Here,two kinds of coccidiostats which including monensin（MON）and madramicin（MD）were selected as reach targets,aiming to develop their monoclonal antibodies（mAb）with high sensitivity,excellent specificity and good affinity.Then the mAb were used as bio-recognition element,and the MOFs-based composites were used as glassy carbon electrode（GCE）modifier or signal probe for constructing electrochemical immunosensors.The immunosensors enabled rapid and sensitive detection of MON and MD by combination of mAb with high specificity and electrochemical analysis with excellent sensitivity.The main research contents of this thesis are as follows:（1）The antigen of MON-BSA and coating antigen MON-OVA were synthesized.Then the antigen was used to immunize BALB/c mice.After cell fusion,a hybridoma cell line of IE10 capable of stably secreting monensin monoclonal antibody（MON mAb）was selected,and the subclass type was IgG1.The optimal coationg concentration for MON-OVA was 1μg/mL,the IC₅₀0 of mAb against MON was 1.02 ng/mL.The cross-reactivity（CR）of the MON mAb towards other compounds including maduramycin,dinitolmide,nicarbazin,sulfadiazine,sulfamethoxazole,sulfathiazole,olaquindox,tetracycline and clenbuterol were all less than 0.05%.（2）The maduramicin antigen and coating antigen were synthesized through EDC/Sulfo-NHS method used bovine serum albumin（BSA）and ovalbumin（OVA）as carrier proteins.A hybridoma cell line of 1B4 capable of stably secreting maduramicin monoclonal antibody（MD mAb）was obtained to prepare MD mAb.The C₅₀0 was calculated to be 1.17 ng/mL based on the established ic-ELISA standard curve of MD mA.The CRs towards MON,dinitolmide,nicarbazin,sulfadiazine,sulfathiazole and quindox were all also less than 0.05%.（3）The Zn/Ni-ZIF-8 metal-organic frameworks（MOFs）were synthesized.Zn/Ni-ZIF-8-800 with high stability was obtained via pyrolysis of the Zn/Ni-ZIF-8 MOFs.Then the Zn/Ni-ZIF-8-800 was combined with graphene to obtain the Zn/Ni-ZIF-8-800@graphene composites which were used to modify the GCE.After that,the Au nanoparticles（AuNPs）were electrodeposited on the modified GCE surface for capturing the MON mAb to construct a label-free electrochemical immunosensor.The preparation process of the immunosensor was investigated by cyclic voltammertry（CV）,and the MON detection procedure was evaluated by differential pulse voltammetry（DPV）.Under the optimal conditions,the linear range was 0.25-100 ng/mL,the detection limit was0.11 ng/mL.In the spiked milk samples,the recoveries of MON were ranged within94.4-112.0%with relative standard deviations（RSDs）of 3.1%-9.2%（n=3）.In addition,the proposed immunosensor was possessed satisfactory reproducibility,acceptable stability and high specificity.（4）An indirect competitive format electrochemical immunosensor was constructed used AuNPs modified GCE as working electrode,and hemin@MOFs/AuPt-Ab₂-HRP/HRP as signal bioprobes for detection of MD residues from egg samples.First,hemin@Fe-MIL-88NH₂ was synthesized by a facile one-step method and further decorated with AuPt nanoparticles for capturing Ab₂-HRP.To eliminate the nonspecific adsorption,the obtained bioconjugates were blocked with horseradish peroxidase（HRP）instead of traditional bovine serum albumin（BSA）to obtain the hemin@MOFs/AuPt-Ab₂-HRP/HRP bioprobes for the reduction of hydrogen peroxide（H₂O₂）.The established immunosensor showed high sensitivity due to the synergistic catalytic effect of hemin@MOFs,AuPt and HRP by a multiple signal amplification system.Under the optimal conditions,the wide detection range of the proposed immunosensor within 0.1-50 ng/mL and a low detection limit of 0.045 ng/mL were achieved.Moreover,the established immunosensor showed high reproducibility,acceptable stability and high sensitivity and selectivity during the detection procedure.