Fluorescence polarization assay to detect the presence of traces of ciprofloxacin

Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).

to directly detect ciprofloxacin residues in commercial diluted milk solution sample. The obtained results show a high sensibility of the assay (1.0 ppb) respect the maximum residual limit (MRL) set by the European Union regulation (100 ppb). Finally, it is worth to note that this method can be transferred into a hand-held device capable to acquire fluorescence polarization changes when ciprofloxacin derivative and anti-ciprofloxacin antibodies interacts.
GlnBP-CPFX conjugate preparation. The GlnBP-CPFX molecules was prepared by conjugation the CPFX to a recombinant glutamine-binding protein (GlnBP) isolated from Escherichia coli 21 . Briefly, the conjugate was synthesized through carbodiimide method described by 15 with slight modifications. Ciprofloxacin 1.0 ml (26.2 mg/ml), was mixed with 1.0 ml of GlnBP (4.5 mg/ml) and EDC (314 mg/ml in 0.01 M Phosphate buffer, pH 5.0). The obtained mix reaction was allowed to occur at 28 °C for 2 h. Dialysis of the reaction mixture was carried out for two days in 0.01 M phosphate buffer, pH 5.0, and the dialyzing buffer was changed twice per day.
Western blotting. Western blotting experiment was performed according to Varriale 19 . In brief, GlnBP, CPFX-GlnBP and CPFX antibodies (4 µg each) were separated by 12% SDS-PAGE and then transferred overnight at 4 °C onto a Polyvinylidene difluoride (PVDF) membrane. After this step, membrane was blocked using TBS containing 5% of milk for 30 min at room temperature, then washed with TBST (three washes for 10 minutes each time.) and incubated with purified mouse monoclonal CPFX antibodies (1:1000) for 1 hr. at 37 °C. After this incubation step, the membrane was washed (three times) and then incubated with goat anti-mouse IgG-HRP conjugate (1:5000) for 1 h at 37 °C. Finally the protein bands developed using ECL. www.nature.com/scientificreports www.nature.com/scientificreports/ Antibody titer determination. To determine the antibody titer according to de Champdoré 19 an ELISA test was performed as described by Varriale 18 with slight modifications. Each well of a 96-well plate was coated by CPFX-GlnBP antigen (0.00625 mg/ml) in bicarbonate buffer (0.05 mol/L, pH 9.6), with different dilutions (1/200, 1/400, 1/800 and 1/1600) at 4 °C overnight. As control, some wells were coated by coating buffer and other wells were coated by GlnBP protein. The wells were washed three times with washing buffer (PBS 0.01 mol/L pH 7.4 containing 0.05% Tween-20; PBST) and after the incubation with 200 μl/well of blocking buffer (0.02 mol/L PBS at pH 7.4 including 2% (w/w) glycine), at 37 °C for 2 h, the plate was rinsed three times. After this step, 100 μl of monoclonal antibodies of different dilutions were incubated in the coated wells at 37 °C for 1 h. The plate was rinsed (three times), goat anti-mouse IgG-HRP antibody (1:5000, 100 μl/well) was added, and the wells were incubated for 1 h at 37 °C. Finally, the enzyme substrate solution (TMB) was added (50 μl /well), and the wells were incubated at 37 °C, then color development was quenched by adding stopping solution H 2 SO 4 (2 mol/L, 50 μl /well) after 10 min. A micro-plate reader was used to measure the absorbance at 450 nm.
100 μl of CPFX-GlnBP (5 mg/ml) in 1 mol/L sodium bicarbonate at pH 8.3 was mixed with the dye CF647 (molar ratio 1:12) and the reaction mixture was kept at room temperature for 1 hour. Separation of CPFX-GlnBP-CF647 from un-reacted probe was done by gel filtration (G25 Sepharose) and extensive dialysis against PBS 0.01 mol/L pH 7.4.

Results
In this study, we describe a fluorescence polarization assay for the detection of the ciprofloxacin. Ciprofloxacin is a fluoroquinolones (FQ) used in the management of pulmonary, urinary and digestive infections in animal livestock. As all antibiotic molecules, ciprofloxacin is a very small molecule (Fig. 1a) unable to elicit an immunological response in an organism. For the assay development, we selected specific commercial monoclonal antibody against the CPFX. In order to characterize the antibody binding features and avoid any cross-reactivity effects, we conjugated the CPFX to a protein carrier. For this purpose, according to Varriale 18 , the CPFX was conjugate to the recombinant GlnBP isolated from E. coli by the well-known conjugation processes using EDC 22,23 . At the end of the conjugation process, SDS-PAGE analysis of the GlnBP and GlnBP-CPFX conjugate was performed to verify the purity of the sample preparations and to evaluate the conjugation effect on the molecular weight of GlnBP (Fig. 1b). The obtained conjugate molecule GlnBP-CPFX, was labeled with a specific fluorescence probe (CF647) and the achieved fluorescence molecule (GlnBP-CPFX-CF647) was used for the development of the competitive fluorescence polarization immunoassay.

Western blotting and ELISA results.
To confirm the specificity of monoclonal antibodies versus the produced GlnBP-CPFX conjugate, western blotting and indirect ELISA tests were performed. In the western blotting experiments, the response was observed against GlnBP-CPFX, while a negative response was showed for GlnBP and BSA (data not shown). On the contrary, an ELISA test was done to evaluate the antibody titer. The value was calculate according to Di Giovanni 24 and as presented in Fig. 1c, it was possible to perform the ELISA test with monoclonal anti-CPFX up to 1 to 1000 dilution.
Competitive ELISA test. A competitive indirect ELISA was performed to set up a sensitive assay for CPFX detection. The plate was coated with fixed amount of GlnBP-CPFX (6,25 μg/μl) and incubated with anti-CPFX in presence of increased concentration of un-labeled CPFX. In Fig. 2  Competitive assay. A competitive fluorescence polarization immune-assay was performed to study the competition between the un-labeled CPFX and GlnBP-CPFX-CF647. For this purpose, samples of anti-CPFX antibody (1000 pM) were incubated in the presence of different amounts of un-labeled CPXF. The data reported in Fig. 4, show the reduction of polarized fluorescence emission at the increasing concentration of un-labelled CPFX. This effect, due by the competition between the anti-CPFX to both GlnBP-CPFX-CF647 and unlabeled CPFX present in solution, allows to detect traces of the CPFX in solution. Figure 4 (inset), reports the change of the fluorescence intensity as a function of CPFX concentration. The presence of a very low concentration of CPFX in solution produces a reduction of the fluorescence emission.
Finally, the developed assay was tested in real food matrices. A competitive experiment was performed diluting increased quantities of CPFX in diluted milk samples. Figure 5 shows the variation of the polarization fluorescence intensity as function of CPFX concentration in diluted milk (1:100). The results show that it is possible to detect less than 1.0 ppb of CPFX directly in the milk solution. This value is lower than the value reported in the EU regulation (100 ppb).

conclusion
Veterinary drugs residues, such as antibiotics, released in animal origin foodstuff have an important consequence for human health and so their detection has become the main aim of food safety control. In this work, we have described the development of a near-infrared fluorescence polarization assay for the detection of the antibiotic CPFX directly in diluted milk solution. The amount of antibiotics in food is rigorously regulated by the European Union and MRLs has been established. This value for ciprofloxacin in milk is 100 ppb (Council Regulation EEC/2377/90).