Development Of Cloxacillin Monoclonal Antibody And Establishment Of Electrochemical Immunoassay

As a semi-syntheticβ-lactam antibiotic,Cloxacillin（CLOX）was used mainly to treat diseases caused by penicillase-producing bacteria including endocarditis and septicemia,leading to widespread application in poultry breeding industry.However,its irrational use may cause excessive CLOX residues in animal-derived food and long-term consumption may endanger human health.To ensure food safety and human health,the relevant authorities in China stipulated that the maximum residue limit（MRL）of CLOX in muscle,fat,liver and kidney was 300μg/kg and MRL in milk was 30μg/kg.Thus,it is of great importance to establish a sensitive,accurate and convenient detection method for CLOX determination.Electrochemical immunosensor has aroused worldwide concern in the fields of food safety detection,environmental monitoring and medicine due to the advantages of simple equipment,convenient operation and high sensitivity.In the paper,CLOX monoclonal antibody（m Ab）was prepared using hybridoma technique.Based on the m Ab,an electrochemical immunosensor was constructed and electrochemical immunoassay method for detecting CLOX was fabricated.Bovine serum albumin（BSA）and ovalbumin（OVA）were respectively conjugated with CLOX by the active ester method to prepare immunogen CLOX-BSA and coating antigen CLOX-OVA.The obtained complete antigens were identified by UV-vis absorption spectrum and SDS-PAGE.The results proved that the complete antigens were successfully synthesized.The polyantiserum titer of immunized mice was determined to be 1:6400 by i ELISA and the half inhibitory concentration(IC₅₀)was 5.37μg/m L by ic ELISA.Three hybridoma cell lines secreting stably CLOX m Ab were obtained by hybridoma technique.The monoclonal cell line 10H10 with the best titer and sensitivity was selected to prepare a mass of monoclonal antibody.The titer of obtained m Ab was 1:5.12×10⁵ and IC₅₀ was 46.77ng/m L.The affinity constant was 2.924×10⁹ L/mol.Moreover,the CLOX m Ab could not react with ampicillin,amoxicillin,piperacillin,methylsulfoxycin and furanone.Based on the GO-PEI-Ag nanomaterials and the prepared CLOX m Ab with high-selectivity and high-sensitivity,an electrochemical immunosensor was constructed to detect CLOX.First,glucose was utilized to reduce silver nanoparticles on the graphene oxide functionalized by polyethyleneimine to prepare GO-PEI-Ag nanocomposites.Then,the obtained GO-PEI-Ag was characterized by SEM,TEM,UV-vis absorption spectrum and XRD.The results indicated that the GO-PEI-Ag nanocomposites were prepared successfully.An electrochemical immunosensor was constructed on the basis of the prepared nanomaterials.Under the optimal conditions,the signal intensity showed linear correlation with the logarithm of CLOX concentration ranging from 0.06 ng/m L to 200 ng/m L,along with the low detection limit of 0.016 ng/m L.In addition,the analytical performance in pork and milk samples was evaluated,showing favorable recovery of 91.33%-105.65%with RSD of2.25%-5.93%.All the results indicated that the sensor had significant potential in the detection of CLOX.