Preparation And Preliminary Application Of Class-specific Monoclonal Antibody For Fluoroquinolones

Fluoroquinolones(FQs) are a group of synthetic antibiotics that are widely used in veteri-nary fields for the treatment and prevention of bacterial infection, which residues in animalfoods are harmful to humans and animals. The detection methods mainly including micro-biological assay, high performance liquid chromatography, liquid chromatography-mass spec-trometry, thin layer chromategraphy, capillaryelectrophoresis, ELISA, Immunosensor, colloidal-gold immunochromatography and electrochemiluminescence. The detect method of immuno-assay for fluoroquinolones have some advantage, but, the key step is preparation of class-specificity antibodies.The objective of this study was to synthesize of artificial antigens for ciprofloxacin. Theaminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten (CPFX-A) and wasproved by (+)ESI-MS spectrum, which was conjugated to bovine serum albumin (BSA) asimmunogen (CPFX-A-BSA) by the N,N’-Dicyclohexylcarbodiimide (DDC) method, and toovalbumin (OVA) as coating antigen(CPFX-A-OVA) by mixed anhydride method, respectively,which were identified by infrared ray (IR) and ultraviolet (UV). The hapten and artificial antigenwere synthesized successfully and antiserum titers of three mice were higher than1:1.28×104, inwhich the titers and IC50of No.2mouse were2.56×104and12.92ng.mL-1,respectively.The objective of this study was to produce class-specific monoclonal antibodys (mAb)against fluoroquinolones, and in order to lay a foundation base for multi-residue detects FQs inanimal foods. Hybridoma lines that secrete mAbs against FQs were selected through cell fusiontechnology and their immunological traits were characterized, which ascites were carried out byin vivo induction method. Three hybridoma cell lines named2H5,3D11,4F4were screenedafter cell fusing, which titers were1:1600,1:1600,1:800in supernatants and1:1.6×106,1:8.2×105,1:8.2×105in ascites, respectively. Cell line named2H5showed good sensitivity andclass-specific toward10FQs, with an IC50values of1.54ng.mL-1～13.60ng.mL-1, lowestdetectable limits (LODs) of0.09ng.mL-1～0.64ng.mL-1and cross-reactivity (CR) of12.6%～111.7%, no cross-reactivity to other compounds was found.The objective of this study was to establish indirect competitive ELISA(icELISA) kit.Using high titer, class-specific monoclonal antibody to detect10FQs in chicken for calculatingrecovery rate and variation coefficient. The data were also compared with that of HPLC, andSPSS17.0software was used to conduct the significant difference analysis. Both of the coefficient variations (CVs) were below10.0%, and no significant difference(P﹥0.05) wasobserved.