Abstract: 【Objective】Fluoroquinolones(FQs) are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study wasto produce class-specific monoclonal antibodies (mAbs) against fluoroquinolones (FQs), establish competitive indirect enzyme linked immunesorbent assay (icELISA), and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】 The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten (CPFX-A) and was proved by (+) ESI-MS spectrum, which was conjugated to bovine serum albumin (BSA) as immunogen (CPFX-A-BSA) by the N,N'-Dicyclohexylcarbodiimide (DDC) method, and to ovalbumin (OVA) as coating antigen(CPFX-A-OVA) by mixed anhydride method, respectively, which were then identified by infrared ray (IR) and ultraviolet (UV). Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and icELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete mAb against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 10⁸ hybridoma cells in vivo, and icELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×10⁴, in which the titer and IC₅₀ of No.2 mouse were1﹕2.56×10⁴ and 12.92 ng·mL^-1. Three hybridoma cell lines named 2H5, 3D11, and 4F4 were screened after 4 times subclone, which titers were 1﹕1 600, 1﹕1 600, and 1﹕800 in supernatants and 1﹕1.6×10⁶, 1﹕8.2×10⁵, and 1﹕8.2×10⁵ in ascites, respectively. The icELISA procedure was optimized at a concentration of CPFX- A-OVA for 1 μg·mL^-1 at 4℃ package overnight by 5% negative serum of pig, monoclonal antibody and GaMIgG-HRP were diluted 1﹕40 000 and 1﹕8 000, respectively. Under the reaction temperature of 37℃, reaction time of standard substance and monoclonal antibody was 15 min, and 25 min after adding GaMIgG-HRP, also 10 min for termination reaction. Cell line named 2H5 showed a good sensitivity and class-specific toward 10 FQs, the linear regression equation was y= -28.022x+56.219，R²=0.9 782，with an IC₅₀ value of 1.67 ng·mL^-1 for ciprofloxacin, 1.82 ng·mL^-1 for norfloxacin, 1.97 ng·mL^-1 for pefloxacin, 1.54 ng·mL^-1 for enrofloxacin, 2.79 ng·mL^-1 for danofloxacin, 3.38 ng·mL^-1 for lomefloxacin, 5.50 ng·mL^-1 for ofloxacin, 4.40 ng·mL^-1 for marbofloxacin, 11.76 ng·mL^-1 for sarafloxacin, 13.60 ng·mL^-1 for difloxacin, and the lowest detectable limits (LODs) of 0.09 ng·mL^-1-0.64 ng·mL^-1 and cross- reactivity (CR) of 12.3%-108.4%, no cross-reactivity to other compounds was found. The recovery ranges of 10 FQs spiked in chicken using icELISA were 80.5%-91.8 %, 85.1%-95.7% for HPLC, both of the coefficient variations (CVs) were below 10.0%, and no significant difference (P＞0.05) was observed.【Conclusion】The high-sensitivity and class-specific mAb against FQs was prepared, which laid a solid foundation for FQs multi-residue detection.

Key words: ciprofloxacin, fluoroquinolones, hybridoma cell lines, class-specific monoclonal antibodies, icELISA