Preparation of Anti-gatifloxacin Antibody and Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Gatifloxacin Residue in Milk

Abstract

A polyclonal anti-gatifloxacin antibody has been prepared, and an indirect competitive enzyme-linked immunosorbent assay (cELISA) was developed on the basis of the antibody prepared for the first time. The antibody shows high sensitivity with an IC50 value of 2.6 ppb and excellent specificity with only a minor cross-reaction with lomefloxacin (3.0%) among common (fluoro)quinolones evaluated in this study. The high specificity of the antibody was explained by the molecular structures of related drugs by comparison with published research. The cELISA test kit developed has a detection limit of 0.05 ppb and could be used as a screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to the detection of milk samples spiked by gatifloxacin. The recovery rates were in the range of 86-106%, whereas the intra- and interassay coefficients of variation were <14.3 and <19.6%, respectively.

Preparation of high-affinity rabbit monoclonal antibodies for ciprofloxacin and development of an indirect competitive ELISA for residues in milk

“…Therefore, more and more organizations have issued maximum residue levels (MRLs) and withdrawal criterion for FQs. According to different food resources and drugs, the MRLs of FQs are set in a range of 30-1500 μg/kg (Lu et al, 2006;Zhao et al, 2007).…”

An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody

“…Huet et al [21] suggested that substituent at position 7 is the governing structural factor determining the binding affinity of fluoroquinolone molecules to anti-norfloxacin antibody and antisarafloxacin antibody. The substituent at position 8 in the structure of gatifloxacin was also believed to be a contribution to the high specificity of anti-gatifloxacin antibody [28]. The importance of substitution at position 1 in fluoroquinolone structures for antibody binding has also been stressed previously [27,30].…”

“…Fortunately, the active carboxylic acid group presented at position 3 in the molecule of norfloxacin (Table 1) enables direct conjugation to a carrier protein [27]. BSA and OVA are among the most commonly used protein carriers [28]. An immunogen NFX-BSA and a coating antigen NFX-OVA were synthesized using the N-hydroxysuccinimide active ester method [25].…”

“…An immunogen NFX-BSA and a coating antigen NFX-OVA were synthesized using the N-hydroxysuccinimide active ester method [25]. UV spectrum was employed to monitor the effectiveness of conjugation reaction [28]. The absorbance for NFX-BSA shows a red-shift maximum at 286 nm compared with the 272 nm maximum for norfloxacin and 273 nm maximum for BSA (Fig.…”

Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine

“…Immunoanalytical techniques have been reported to detect GAT by an enzyme-linked immunosorbent assay (ELISA) using ultraviolet (UV) or fluorescence absorbance as a quantitative parameter (Santoro et al, 2006;Zhao et al, 2007). In recent years, electrochemical immunoassays have attracted attention because of their excellent sensitivity, simplicity, inexpensive construction, suitability for mass production, and their variety of labels, including enzymes and electro-active molecules.…”