Heterologous strategy enhancing the sensitivity of the fluorescence polarization immunoassay of clinafloxacin in goat milk
Jiahong Chen a , Ilya A.Shanin b,c , Shuwei Lv a, Qiang Wang d, Chuanbin Maoe, Zhenlin Xu a, Yuanming Sun a, Qing Wu a, Sergei A. Eremin b,c*, Hongtao Lei a*
a
Guangdong Provincial Key Laboratory of Food Quality and Safety / Guangdong Provincial Engineering
& Technique Research Centre of Food Safety Detection and Risk Assessment, South China Agricultural University, Guangzhou 510642, China b
Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie gory 1, Building 3, 119991
Moscow, Russia c
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia
d
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300,
China e
Department of Chemistry & Biochemistry, Stephenson Life Sciences Research Center, University of
Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA
*
Corresponding authors.
Hongtao Lei, Tel. +86 20 8528 3448, Fax. +86 20 8528 0270, E-mail: [email protected]
Sergei A. Eremin, Tel. +7 495 9394192, Fax. +7 495 9392742, Email: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7228
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Abstract BACKGROUND: Clinafloxacin is used for the treatment of disease in food-producing animals, e.g., Brucella melitensis, which often occurs in goats; however, the clinafloxacin residue in goat milk may harm human health and result in the development of drug-resistant bacterial strains or allergies. Despite this, there is not a rapid, sensitive and accurate analytical method in goat milk for a rapid screening or monitoring purpose. RESULTS: One homologous and five heterologous tracers were designed and compared for fluorescence polarization immunoassay(FPIA) optimization. Based on the combination of a heterologous tracer (PAZ-FITC, synthesized with pazufloxacin and FITC) and the antibody against clinafloxacin, a highly sensitive FPIA was established for the detection of clinafloxacin residue in goat milk for the first time. The IC50 value was 29.3 μg/L for clinafloxacin in the heterologous format, 6 times lower than that of the combination of the homologous tracers and the antibody. The recoveries ranged from 86.8% to 104.5%, with the relative standard deviation ranging from 4.1% to 7.2%. Validation by high-performance liquid chromatography (HPLC) confirmed that the results obtained from the proposed FPIA were in agreement with those of HPLC. CONCLUSION: This proposed heterologous strategy for enhanced FPIA is sensitive and rapid enough for the high-throughput detection of clinafloxacin residue in goat milk. Keywords: heterologous; clinafloxacin; fluorescence polarization immunoassay; goat milk
ABBREVIATIONS USED FPIA,
fluorescence
polarization
immunoassay;
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HPLC,
high-performance
liquid
chromatography; CLI, clinafloxacin; FQs, fluoroquinolones; MRLs, maximum residue limits; KFDA, Korea Food and Drug Administration; EU, European Union; CE, capillary electrophoresis methods; RP-HPLC, reversed phase high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate isomer I; anti-CLI, anti-clinafloxacin; TLC, thin-layer chromatography; PAZ, pazufloxacin; PIP, pipemidic acid; ENO, enoxacin; NOR, norfloxacin; CIP, ciprofloxacin; BB, borate buffer; PBS, phosphate buffer; LOD, limit of detection; CR, Cross-reactivity; RSD, relative standard deviation.
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INTRODUCTION Clinafloxacin (CLI) (Figure 1) is a type of broad-spectrum halogenated fluoroquinolone (FQ), which is more active against most Gram-positive bacteria, Gram-negative bacteria and anaerobic organisms. In recent years, clinafloxacin has been used for the treatment of disease in food-producing animals
1, 2
, e.g., Brucella melitensis, which occurs mainly in goats.
However, through the food chain, clinafloxacin residue may persist in humans, resulting in the development of drug-resistant bacterial strains or allergies. Goat milk is widely and popularly produced and consumed in China, Russia, France, Greece, Italy and Spain, etc3. The problem of antibiotic residue in goat milk has already become a concern worldwide4. To protect consumers from potentially contaminated goat milk, China has set the maximum residue limits (MRLs) of FQs at 100 μg/L5. The Korea Food and Drug Administration (KFDA) set the maximum amount of veterinary drugs in goat milk as 0.03 mg/kg6. The European Union (EU) has set the MRLs of most FQs at 100 μg/kg7. Many different techniques have been described for the determination of clinafloxacin in biological matrices, including high-performance liquid chromatography (HPLC)8, capillary electrophoresis methods (CE)9, and reversed phase high-performance liquid chromatography (RP-HPLC)10. Although chromatographic methods provide accurate and sensitive analysis, they are laborious, time consuming and low throughput. Compared with instrumental methods, immunochemical methods such as enzyme-linked immunosorbent assay (ELISA), which have been successfully used for both official and laboratory purposes, have adequate sensitivity and are high throughput11-13. Fluorescence polarization immunoassay (FPIA)14, 15 has advantages over ELISA in that the assay is time saving, has fewer steps, provides
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convenient manipulation and can be easily automated. However, due to the complexity of tracer design and synthesis, trace tracer separation and assay optimization significantly differed from the traditional solid phase immunoassays such as ELISA, radioimmunoassay or immunochromatography. It is still an important challenge to develop a highly sensitive FPIA for a bioanalytical chemist. To our knowledge, there has not yet been an FPIA developed method for the rapid and sensitive detection of clinafloxacin residue in goat milk. In this study, one structurally homologous tracer and five structurally heterologous tracers were designed and synthesized to improve the sensitivity (Figure 1). Compared to the usual homologous immunoassay format, herein, the sensitivity of the established FPIA was dramatically
enhanced
based
on
a
heterologous
tracer,
(S)-10-(1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl)thioureido)cyclopropyl )-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid. HPLC confirmation validated the reliability of the proposed FPIA for use as an ideal rapid-screening and high-throughput tool. This is the first FPIA with a structurally heterologous tracer to improve the sensitivity for the detection of clinafloxacin present in goat milk.
MATERIALS AND METHODS Reagents and instrumentation Fluorescein isothiocyanate isomer I (FITC) and triethylamine were purchased from Sigma (St. Louis, MO, USA). Anti-clinafloxacin (anti-CLI) polyclonal antibody was generated previously by Guangdong Provincial Key Laboratory of Food Quality and Safety.
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Silica gel 60 aluminum sheets (type GF254, layer thickness 0.25 mm) for thin-layer chromatography (TLC) were purchased from Merck (Darmstadt, Germany). The analytical standards (clinafloxacin, pazufloxacin (PAZ), pipemidic acid (PIP), enoxacin (ENO), norfloxacin (NOR), ciprofloxacin (CIP)) were donated by the veterinary college of South China Agricultural University (Guangdong, China). Goat milk was obtained from a local farm (Moscow, Russia) and was assumed as initially FQ free. Other reagents and organic solvents were analytical grade. Borate buffer (BB, 0.05 mol/L, pH 8.0) with 0.1% Tween-20 and 0.1% methanol (BBTM) was used as the working buffer for all FPIA experiments. Phosphate buffer (PBS, 0.1 mol/L, pH 7.4) with 2% methanol (PBSM) was used as the working buffer for aqueous standard solutions of the analytes. FPIA was conducted using a TDX/FLX Analyzer (Abbott Laboratories, Irving, TX USA) in semi-automatic PhotoCheck mode. TDX/FLX glass cuvettes (up to 10 in one run) were loaded into the special carousel, followed by the measurement of polarization (in mP units) and the intensity (in conventional units) of fluorescence.
Synthesis of fluorescein-labeled tracers The FITC-labeled pazufloxacin (tracer PAZ-FITC) was synthesized as below. Briefly, 2.0 mg pazufloxacin was dissolved in methanol (1.0 mL). Then, 160 μL FITC solution (10 mg FITC was dissolved in 0.8 mL methanol) and 0.2 mL triethylamine were added dropwise to the solution. After stirring at room temperature overnight in the dark16, small portions of the reaction mixture were first separated by TLC using CHCl3: methanol= 1:1(V:V) as the
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eluent. The plates were dried and redeveloped with CHCl3: methanol: ammonia= 5:2:0.4 (V: V: V). The major yellow bands of varying Rf values were collected, eluted with 0.5 mL methanol and stored at −20℃ under the conditions of protection from light. The tracer concentration was evaluated as in the previously reported method17. The working tracer concentration was chosen as the fluorescence intensity for free tracer which have fluorescence intensity 10 times higher than buffer at approximately 2000-2500 units. The other five fluorescein-labeled tracers (CLI-FITC, PIP-FITC, ENO-FITC, NOR-FITC, CIP-FITC) were synthesized as in the procedure above (Figure 1). Subsequently, to obtain the optimal tracer with sufficient binding capability to the anti-CLI, each tracer was diluted and mixed with a fixed antibody diluted solution. A series of calibration curves was constructed and the binding activity evaluation was completed by comparing three parameters (δmP, IC50 and δmP/IC50). Herein, δmP means the difference between the maximal and minimal fluorescence polarization signals. Lower IC50 values and higher values of δmP and δmP/IC50 indicate a more sensitive assay18.
Fluorescence polarization immunoassay procedure 50 μL clinafloxacin standard solution was mixed with 500 μL tracer-diluted solution, and 500 μL of the diluted antibody was used to construct a series of calibration curves for the selection of the optimal antibody dilution according to three parameters (δmP, IC50 and δmP/IC50) 18. The clinafloxacin standard solution (50 μL), at concentrations from 0.1 μg/L to 100,000 μg/L (0.1 g/L), was mixed with the diluted tracer (500 μL) and the diluted antibody (500 μL)
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to construct the competitive calibration curves. The mP values were plotted against the analyte concentration, and a four-parameter equation was used to fit the experimental sigmoidal curve using Origin 8.5 for Windows19-21: y= (A-D)/ [1+(x/C)B]+D In the equation, A is the maximal mP, D is the minimal mP, C is the clinafloxacin concentration at 50% of tracer binding (IC50) and B is the slope of the curve. The working range represents the standard concentration at 20%–80% of tracer binding (IC20－IC80)22, the limit of detection (LOD) was defined as the standard concentration at 10% of tracer binding22, and the limit of quantification (LOQ) was set as the standard concentration of 20 % tracer binding23-25.
Specificity The specificity of the FPIA was determined using 6 kinds of various FQs under optimized FPIA conditions. Cross-reactivity (CR) was calculated according to the following equation, where IC50 is the concentration at which 50% of the anti-CLI is bound to the analyte. CR%= [IC50 (clinafloxacin)/IC50 (structurally related compounds)] ×100%
Recovery To evaluate the recovery of the developed FPIA, goat milk was spiked with clinafloxacin and the recoveries were determined by FPIA. Three concentrations of the clinafloxacin standard were added to the goat milk samples. Saturated ammonium sulfate
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solution was added to each goat milk solution (V: V=1:1) to precipitate protein. Then, the samples were centrifuged for 10 min (13,000 r/min, 4 °C) to remove the fat from the goat milk. The lower liquid nonfat phase was diluted 1:4 in borate buffer before being used in FPIA. Blank samples were prepared as described above but were not spiked with analyte. Finally, the clinafloxacin concentration in spiked samples was detected by the established FPIA.
HPLC confirmation Goat milk samples for HPLC analysis were prepared according to the method described by Chinese National Standard (GB 29692-2013) 26. Briefly, homogenized goat milk samples (2 mL), spiked with different concentrations of clinafloxacin, were added to 100 μL phosphoric acid and 4 mL acetonitrile and stirred for 5 min on a shaker. After centrifugation (10,000×g, 10 min), the supernatant was transferred into a centrifuge tube. Then, 5 mL n-hexane was added to the supernatant and stirred for 1 min. After several minutes of standing, the subnatant was transferred into a 25 mL specimen bottle. The residue was again extracted with 4 mL acetonitrile. Both extraction solutions were mixed and evaporated to dryness at 50 ℃. The residue was redissolved in 1 mL mobile phase (0.05 mol/L phosphoric acid-triethylamine (pH 2.4) and acetonitrile (90:10, v/v)), and the solution was filtered (0.22 μm) and transferred to an HPLC sample vial ready for analysis. HPLC analysis was performed by a Waters E2695 HPLC with a fluorometric detector (model 2475). The excitation wavelength and emission wavelength were 290 nm and 490 nm, respectively. A C18 column (150 × 4.6 mm, 5 μm particle size; Agilent) was used as the
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stationary phase. The flow rate was 1 mL/min, and the column temperature was 30℃. The measured results were compared with the FPIA results. The linear correlation between the results of two methods was verified by the Student's t-test.
RESULTS AND DISCUSSION Effect of tracer structure on FPIA sensitivity The tracer structure could greatly influence the immunoassay performance
27
. Among
the obtained six tracers (the homologous one (CLI-FITC) and the heterologous ones (PAZ-FITC, PIP-FITC, CIP-FITC, ENO-FITC and NOR-FITC)), compared with the negative one, each tracer showed δmP value from 90 to 199 mP, which could demonstrate sufficient binding capability with the employed antibody against clinafloxacin (Figure 2)28-30. Thus, the working concentration of these tracers in FPIA was approximately 4 nmol/L, as in the previously reported method17. According to the antibody dilution curve, the concentration demonstrating 70% binding with the antibody was selected as the working concentration31, 32. Herein, the 1/200 dilution titer was selected as the working concentration of antibody, and thus, the calibration curve for clinafloxacin with different tracers was plotted (Table 1). Compared to the homologous tracer CLI-FITC, all four of the heterologous tracers (PAZ-FITC, PIP-FITC, ENO-FITC and NOR-FITC) demonstrated a significantly lower IC50 (2-6 times lower). PAZ-FITC, exhibited the highest δmP (150), the lower IC50 (29.3 μg/L) and the maximal δmP/IC50 (5.12); its IC50 showed it had 6 times higher sensitivity than the homologous tracer CLI-FITC. This could be ascribed that a heterologous tracer usually results in weaker recognition by the antibody than
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the recognition to the analyte. The analyte mimics the immunizing hapten (used for the immunogen) more closely than the tracer hapten33, 34, as a result, the affinity of the analyte to the antibody has likely been relatively improved 35 and the detection sensitivity was enhanced accordingly. The optimized, typed calibration curve for clinafloxacin is shown in Figure 3. The LOD (10% inhibition concentration) was 4.1 μg/L, the LOQ (20% inhibition concentration) was 8.5 μg/L, and the working range (20%–80% inhibition concentration) was 8.5－100.5 μg/L. The performance assay of this proposed FPIA could meet the requirements for the FQ residue limitation in China, Korea and the EU5, 6.
Specificity The cross-reactivities (CRs) of 6 kinds of FQs were determined using the developed FPIA with clinafloxacin as the reference compound (CR=100%) (Table 2). The CRs for pazufloxacin and ciprofloxacin were 26.6% and 32.5%, respectively, while the others were lower. Comparing the structures of clinafloxacin and ciprofloxacin, a primary amine at position 7 for clinafloxacin was changed to a dinonyl amine in ciprofloxacin. The CR decreased from 100% for clinafloxacin to 32.5% for ciprofloxacin, implying that the amine group was likely a key structural factor for antibody recognition. In addition, although enoxacin differs from pipemidic acid in structure only by the group at position 6, the antibody showed a higher recognition to enoxacin (CR, 10.9%) than pipemidic acid (CR, 4.7%), suggesting that position 6 is also an important site for antibody recognition. Pazufloxacin showed a CR of 26.6% to the antibody, however, to date, it was a human
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used antibiotic to treat infections of the respiratory tract, urinary-reproductive tract and post-operative infections36-38, as no country has permitted it to be used in animal reproduction and treatment. Therefore, pazufloxacin will seldom occur in goat milk. Ciprofloxacin is a broad-spectrum FQ, and it was often reported to exhibit broad CR with other FQs due to the shared common structure of other FQ drugs39, 40. It seems that the high CR from ciprofloxacin is not usually evitable in the immunoassay of FQ drugs26, 27 but a statutory confirmatory method is usually still needed to exclude the false positive after a screening assay41. For a rapid screening purpose, the FPIA with low CR is acceptable considering the speed and convenience of an FPIA42, 43.
Recovery The average recovery of clinafloxacin from fortified goat milk was 96.6%, ranging from 86.8%–104.5% (Table 3), and the average relative standard deviation (RSD) was 5.3%, ranging from 4.1%–7.2%. This indicated that the recovery and reproducibility of the developed FPIA were satisfactory for a rapid screening analysis.
HPLC confirmation The analytical results obtained with the FPIA and HPLC methods showed good correlation (R2 = 0.9665) (Figure 4). The P value of less than 0.01 by Student's t-test was considered significant positive correlation between the results of two methods.
This
suggested that the developed heterologous strategy-based FPIA method could be used as a rapid screening and high-throughput method for monitoring clinafloxacin residue in goat
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milk samples.
CONCLUSIONS Six tracers with different structures were designed to optimize the assay performance. The heterologous structure strategy significantly enhanced the sensitivity up to 6 times compared to the usual homologous assay format. The established FPIA was successfully used for milk samples with excellent specificity, recovery and relative standard deviation. HPLC confirmation also validated the reliability of the developed FPIA. In conclusion, the proposed FPIA could be considered a highly sensitive and rapid high-throughput method for monitoring clinafloxacin residue in goat milk.
ACKNOWLEDGEMENTS This work was supported by Guangdong Planed Program in Science and Technology (cgzhzd0808, 2013B051000072, 2012B090600005), the PhD Programs Foundation of Ministry of Education of China (20114404130002), Guangdong Natural Science Foundation (S2013030013338), Natural Science Foundation of China (U1301214), the Russian State Targeted Program ‘Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020’ (contract 14.613.21.0028, unique identifier of applied research: RFMEFI61314X0028) and the Russian Foundation for Basic Research grants 14-03-00753, 13-03-93000 and 12-03-92105.
REFERENCES
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1 Sweeney MT, Quesnell R, Tiwari R, LeMay M and Watts JL, In vitro activity and rodent efficacy of clinafloxacin for bovine and swine respiratory disease. Front Microbiol 4:154 (2013). 2 García-Rodríguez JA, Sanchez JG, Trujillano I, Sánchez EG, Garcia MG and Fresnadillo M, Susceptibilities of Brucella melitensis isolates to clinafloxacin and four other new fluoroquinolones. Antimicrob Agents Ch 39:1194-1195 (1995). 3 Pirisi A, Lauret A, Dubeuf JP. Basic and incentive payments for goat and sheep milk in relation to quality. Small Ruminant Res 68:167-178(2007). 4
Liu YZ, Jiang LQ and Xi HP, Research progress of determination method for fluoroquinolones residue in milk and dairy products. Food Ind 34:185-188 (2013).
5 Ministry of Agriculture of the People’s Republic of China, Maximum residue limits for veterinary drugs in animal products. (2002). 6 Korea Food and Drug Administration, Korea residue limits of veterinary drugs in food. (2008). 7 Official Journal of the European Communities. Commision Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. (2009). 8 Zhou MH, Yu YQ, Duan GL, Cheng WB, Liu X, Cao Q and Zhang Y, Determination of clinafloxacin in rat plasma by HPLC. Chin Pharm Anal 21:270-273 (2001). 9 Navalón A, Araujo L, Prieto A and Vı́lchez JL, Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis. J Chromatogr B 772:65-72 (2002). 10 Xu Q, Zhang JX, Shi NN and Zhou XX, Determination of clinafloxacin in plasma of rats
This article is protected by copyright. All rights reserved
by RP-HPLC. Chin J Pharm 31:316-318 (2000). 11 Wang WS, Shih CW, Hung SW, Ling YF, Tu CY, Chen BR, Tsou LT and Ho SP, Development and application of the enzyme-linked immunosorbent assay residual detection kits for fluoroquinolones. Taiwan Vet J 32:391-403 (2006). 12 Wen K, Nolke G, Schillberg S, Wang ZH, Zhang SX, Wu CM, Jiang, HY, Meng H and Shen JZ, Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones. Anal Bioanal Chem 403:2771-2783 (2012). 13 Kuhlhoff S and Diehl Y, Rapid screening test for determination of fluoroquinolones in commercial poultry meat. Dtsch Lebensm-Rundsch 101:384-388 (2005). 14 Eremin SA. Fluorescence polarisation immunoassays for determination of pesticides and biologically active compounds in food safety and environmental monitoring. Food Technol Biotech 36:235-244(1998). 15 Eremin SA. Polarization fluoroimmunoassay for rapid, specific detection of pesticides. Chapter 16 In: "ACS Symposium Series No.586, Immunoanalysis of Agrochemicals: Emerging Technologies" (Eds. Judd O. Nelson, Alexander E. Karu, and Rosie Wong). American Chemical Society, Washington, DC 223-234 (1995). 16 Yakovleva JN, Lobanova AY, Shutaleva EA, Kourkina MA, Mart’ianov AA, Zherdev AV, Dzantiev BB and Eremin SA, Express detection of nonylphenol in water samples by fluorescence polarization immunoassay. Anal Bioanal Chem 378:634-641 (2004). 17 Hatzidakis GI, Tsatsakis AM, Krambovitis EK, Spyros A, Eremin SA. Use of L-lysine fluorescence derivatives as tracers to enhance the performance of polarization
This article is protected by copyright. All rights reserved
fluoroimmunoassays. A study using two herbicides as model antigens. Anal Chem 74:2513-2521(2002). 18 Lei HT, Xue G, Yu CF, Haughey SA, Eremin SA, Sun YM, Wang ZH, Xu ZL, Wang H and Shen YD, Fluorescence polarization as a tool for the detection of a widely used herbicide, butachlor, in polluted waters. Anal Methods 3:2334-2340 (2011). 19
Oberleitner L, Grandke J, Mallwitz F, Resch-Genger U, Garbe LA, Schneider RJ. Fluorescence Polarization Immunoassays for the Quantification of Caffeine in Beverages. J Agr Food Chem 62:2337-2343(2014).
20
Eremin SA, Ryabova IA, Yakovleva JN, Yazynina EV, Zherdev AV, Dzantiev BB. Development of a rapid, specific fluorescence polarization immunoassay for the herbicide chlorsulfuron. Anal Chim Acta.468:229-236(2002).
21
Benito-Peña E, Moreno-Bondi MC, Orellana G, Maquieira Á, van Amerongen A. Development of a novel and automated fluorescent immunoassay for the analysis of β-lactam antibiotics. J Agr Food Chem 53:6635-6642(2005).
22 Murtazina NR, Eremin SA, Mozoleva OV, Everest SJ, Jim Brown A and Jackman R, Fluorescent polarization immunoassay for sulphadiazine using a high specificity antibody. Int J Food Sci Tech 39:879-889 (2004). 23
Parra J, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A. Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays. Anal Chim Acta 715:105-112(2012).
24
Suarez-Pantaleon C, Wichers J, Abad-Somovilla A, van Amerongen A, Abad-Fuentes
This article is protected by copyright. All rights reserved
A. Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron. Biosens Bioelectron 42:170-176(2013). 25
Garcia-Fernandez J, Trapiella-Alfonso L, Costa-Fernandez JM, Pereiro R, Sanz-Medel A. A Quantum Dot-Based Immunoassay for Screening of Tetracyclines in Bovine Muscle. J Agric Food Chem 62:1733-1740(2014).
26 Ministry of Agriculture of the People’s Republic of China, National Health and Family Planning Commission of the People’s Republic of China, Determination of quinolones residues in milk by high performance liquid chromatographic method (GB 29692-2013). (2013). 27 Choi EH, Kim DM, Choi SW, Eremin SA and Chun HS, Optimisation and validation of a fluorescence polarisation immunoassay for rapid detection of zearalenone in corn. Int J Food Sci Tech 46:2173-2181 (2011). 28
Wang ZH, Cheng LL, Shi WM, Zhang SX, Shen JZ. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies. Sci China Chem 53:553-555(2010).
29 Mi TJ, Wang ZH, Eremin SA, Shen JZ, Zhang SX. Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay. J Agric Food Chem 61:9347-9355(2013). 30
Zhang J, Wang ZH, Mi TJ, Wenren LQ, Wen K. A homogeneous fluorescence polarization immunoassay for the determination of cephalexin and cefadroxil in milk. Food Anal Methods 7:879-86(2014).
This article is protected by copyright. All rights reserved
31
Ren LL, Meng M, Wang P, Xu ZH, Eremin SA, Zhao JH, Yin YM, Xi RM. Determination of sodium benzoate in food products by fluorescence polarization immunoassay. Talanta.121:136-143(2014).
32
Mi TJ, Wang ZH, Eremin SA, Shen JZ, Zhang SX. Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay. J Agric Food Chem. 61:9347-9355(2013).
33
Wang ZH, Zhang SX, Shen JZ and Eremin SA. Analysis of sulfamethazine by fluorescence polarization immunoassay. Chin J Anal Chem 35:819-824(2007).
34
Xu ZL, Wang Q, Lei HT, Eremin SA, Shen YD, Wang H, Beier RC, Yang JY, Maksimova KA and Sun YM. A simple, rapid and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples. Anal Chim Acta 708:123-129(2011).
35 Zhang Q, Wang LB, Ahn KC, Sun Q, Hu BS, Wang J and Liu FQ, Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion. Anal Chim Acta 596:303-311(2007). 36
Nomura T, Hirai K, Yamasaki M, Inoue T, Takahashi M, Kawashima T, Sato F and Mimata H. Efficacy of prophylactic single-dose therapy using fluoroquinolone for prostate brachytherapy. Jpn J Radiol 30:317-322(2012).
37 Luo YL, Mo BW, Yin YS, Zhang ZB, Yan SF, Yue HM and Cao XL. A multiple center randomized controlled clinical study of pazufloxacin mesylate for injection in treatment of patients with acute bacterial infections. Chin J New Drugs Clin Rem 32:534-538(2013).
This article is protected by copyright. All rights reserved
38 Uegami S, Ikawa K, Ohge H, Nakashima A, Shigemoto N, Morikawa N, Murakami Y, Sueda T, Uegami S, Ohge H, Nakashima A, Shigemoto N, Murakami Y and Sueda T. Pharmacokinetics
and
pharmacodynamic
target
attainment
of
intravenous
pazufloxacin in the bile of patients undergoing biliary pancreatic surgery. J Chemother 26:287-292(2014). 39 Duan JH, Yuan ZH. Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues. J Agric Food Chem 49:1087-1089(2001). 40 Jiang JQ, Zhang HT, An ZX, Xu ZY, Yang XF, Huang HG and Wang ZL. Development of an heterologous immunoassay for ciprofloxacin residue in milk. Phys Procedia 25:1829-1836(2012).
41 Official Journal of the European Communities. Commission Decision of 12 August 2002 Implementing the Council Directive 96/23/EC Concerning the Performance of Analytical Methods and the Interpretation of Results (2002/657/EC). (2002). 42 Shim WB, Yakovleva ME, Kim KY, Nam BR, Vylegzhanina ES, Komarov AA, Eremin SA and Chung DH. Development of Fluorescence Polarization Immunoassay for the Rapid Detection of 6-Chloronicotinic Acid: Main Metabolite of Neonicotinoid Insecticides. J Agric Food Chem 57:791-796(2009). 43 Li M, Liu XF, Hua XD, Yin W, Fang QK and Wang MH. Fluorescence polarization immunoassay for highly efficient detection of clothianidin in agricultural samples. Anal Methods 6:6541-6547(2014).
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Figure captions
Figure 1 Schematic routes for the synthesis of 6 kinds of (fluoro)quinolone tracers. Figure 2 Dilution curves of the anti-clinafloxacin antibody. Figure 3 FPIA calibration curve for clinafloxacin (n=3). Figure 4 Correlation between FPIA and HPLC for spiked goat milk (n=3).
Figure 1 Schematic routes for the synthesis of 6 kinds of (fluoro)quinolone tracers.
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PAZ-FITC CLI-FITC PIP-FITC ENR-FITC NOR-FITC CIP-FITC Negative
mP
300
200
100
0
100
1000
10000
Dilution curve of antibody
Figure 2 Dilution curves of the anti-clinafloxacin antibody.
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Table 1 Analytical characteristics of FPIA with homologous and heterologous tracers. Antibody
anti-CLI
Titer of antibody
Tracer
δmP
1/200
PAZ-FITC CLI-FITC PIP-FITC ENO-FITC NOR-FITC CIP-FITC
150 120 55 40 45 55
IC50 (μg/L) 29.3 181.0 31.3 37.6 27.3 113.1
δmP/IC50 5.12 0.66 1.76 1.06 1.65 0.49
IC20－IC80 (μg/L) 8.5－100.5 39.0－837.4 8.1－120.5 8.1－174.4 7.7－96.7 32.7－391.3
LOD (μg/L) 4.1 15.9 3.7 3.3 3.7 15.8
δmP means the response span between the maximal and minimal fluorescence polarization signals 18.
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Table 2 Cross-reactivity (CR) for various fluoroquinolones with clinafloxacin No 1 2 3 4 5 6
Compound Clinafloxacin Pazufloxacin Pipemidic acid Enoxacin Norfloxacin Ciprofloxacin
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CR IC50 (nmol/mL) (%) 0.07 100.0 0.27 26.6 1.54 4.7 0.60 10.9 0.67 10.9 0.22 32.5
Table 3 Recovery of clinafloxacin from spiked goat milk samples detected by FPIA (n=3) Spiked level (μg/L) 100 250 500
Found level (μg/L)
Recovery (%, n=3)
99.0±4.0 249.1±11.8 455.9±32.8
99.0±4.0 99.6±4.7 91.2±6.6
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Mean recovery (%)
RSD (%)
Mean RSD (%)
96.6
4.1 4.8 7.2
5.3
mP
220
PAZ-FITC, 4 nmol/L anti-CLI, 1/200 IC50=29.3 μg/L
200 180
LOD=4.1 μg/L Working range=8.5−100.5 μg/L Equation: y=151.5 / [1+x/29.3]1.1+53.9 R2=0.99985
160 140 120
O
100 80
HCl H2 N
O
F
OH
N
N Cl
60 40 10-2 10-1
100
101
102
103
104
105
106
Concentration of clinafloxacin,μg/L
Figure 3 FPIA calibration curve for clinafloxacin (n=3).
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HPLC (μg/L)
400
300
200
y=0.87x-13.31 R2=0.9665
100
100
200
300
400
500
FPIA (μg/L)
Figure 4 Correlation between FPIA and HPLC for spiked goat milk (n=3).
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Jiahong Chen a , Ilya A.Shanin b,c , Shuwei Lv a, Qiang Wang d, Chuanbin Maoe, Zhenlin Xu a, Yuanming Sun a, Qing Wu a, Sergei A. Eremin b,c*, Hongtao Lei a*
a
Guangdong Provincial Key Laboratory of Food Quality and Safety / Guangdong Provincial Engineering
& Technique Research Centre of Food Safety Detection and Risk Assessment, South China Agricultural University, Guangzhou 510642, China b
Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie gory 1, Building 3, 119991
Moscow, Russia c
A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow 119071, Russia
d
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300,
China e
Department of Chemistry & Biochemistry, Stephenson Life Sciences Research Center, University of
Oklahoma, 101 Stephenson Parkway, Norman, OK 73019, USA
*
Corresponding authors.
Hongtao Lei, Tel. +86 20 8528 3448, Fax. +86 20 8528 0270, E-mail: [email protected]
Sergei A. Eremin, Tel. +7 495 9394192, Fax. +7 495 9392742, Email: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7228
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Abstract BACKGROUND: Clinafloxacin is used for the treatment of disease in food-producing animals, e.g., Brucella melitensis, which often occurs in goats; however, the clinafloxacin residue in goat milk may harm human health and result in the development of drug-resistant bacterial strains or allergies. Despite this, there is not a rapid, sensitive and accurate analytical method in goat milk for a rapid screening or monitoring purpose. RESULTS: One homologous and five heterologous tracers were designed and compared for fluorescence polarization immunoassay(FPIA) optimization. Based on the combination of a heterologous tracer (PAZ-FITC, synthesized with pazufloxacin and FITC) and the antibody against clinafloxacin, a highly sensitive FPIA was established for the detection of clinafloxacin residue in goat milk for the first time. The IC50 value was 29.3 μg/L for clinafloxacin in the heterologous format, 6 times lower than that of the combination of the homologous tracers and the antibody. The recoveries ranged from 86.8% to 104.5%, with the relative standard deviation ranging from 4.1% to 7.2%. Validation by high-performance liquid chromatography (HPLC) confirmed that the results obtained from the proposed FPIA were in agreement with those of HPLC. CONCLUSION: This proposed heterologous strategy for enhanced FPIA is sensitive and rapid enough for the high-throughput detection of clinafloxacin residue in goat milk. Keywords: heterologous; clinafloxacin; fluorescence polarization immunoassay; goat milk
ABBREVIATIONS USED FPIA,
fluorescence
polarization
immunoassay;
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HPLC,
high-performance
liquid
chromatography; CLI, clinafloxacin; FQs, fluoroquinolones; MRLs, maximum residue limits; KFDA, Korea Food and Drug Administration; EU, European Union; CE, capillary electrophoresis methods; RP-HPLC, reversed phase high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate isomer I; anti-CLI, anti-clinafloxacin; TLC, thin-layer chromatography; PAZ, pazufloxacin; PIP, pipemidic acid; ENO, enoxacin; NOR, norfloxacin; CIP, ciprofloxacin; BB, borate buffer; PBS, phosphate buffer; LOD, limit of detection; CR, Cross-reactivity; RSD, relative standard deviation.
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INTRODUCTION Clinafloxacin (CLI) (Figure 1) is a type of broad-spectrum halogenated fluoroquinolone (FQ), which is more active against most Gram-positive bacteria, Gram-negative bacteria and anaerobic organisms. In recent years, clinafloxacin has been used for the treatment of disease in food-producing animals
1, 2
, e.g., Brucella melitensis, which occurs mainly in goats.
However, through the food chain, clinafloxacin residue may persist in humans, resulting in the development of drug-resistant bacterial strains or allergies. Goat milk is widely and popularly produced and consumed in China, Russia, France, Greece, Italy and Spain, etc3. The problem of antibiotic residue in goat milk has already become a concern worldwide4. To protect consumers from potentially contaminated goat milk, China has set the maximum residue limits (MRLs) of FQs at 100 μg/L5. The Korea Food and Drug Administration (KFDA) set the maximum amount of veterinary drugs in goat milk as 0.03 mg/kg6. The European Union (EU) has set the MRLs of most FQs at 100 μg/kg7. Many different techniques have been described for the determination of clinafloxacin in biological matrices, including high-performance liquid chromatography (HPLC)8, capillary electrophoresis methods (CE)9, and reversed phase high-performance liquid chromatography (RP-HPLC)10. Although chromatographic methods provide accurate and sensitive analysis, they are laborious, time consuming and low throughput. Compared with instrumental methods, immunochemical methods such as enzyme-linked immunosorbent assay (ELISA), which have been successfully used for both official and laboratory purposes, have adequate sensitivity and are high throughput11-13. Fluorescence polarization immunoassay (FPIA)14, 15 has advantages over ELISA in that the assay is time saving, has fewer steps, provides
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convenient manipulation and can be easily automated. However, due to the complexity of tracer design and synthesis, trace tracer separation and assay optimization significantly differed from the traditional solid phase immunoassays such as ELISA, radioimmunoassay or immunochromatography. It is still an important challenge to develop a highly sensitive FPIA for a bioanalytical chemist. To our knowledge, there has not yet been an FPIA developed method for the rapid and sensitive detection of clinafloxacin residue in goat milk. In this study, one structurally homologous tracer and five structurally heterologous tracers were designed and synthesized to improve the sensitivity (Figure 1). Compared to the usual homologous immunoassay format, herein, the sensitivity of the established FPIA was dramatically
enhanced
based
on
a
heterologous
tracer,
(S)-10-(1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl)thioureido)cyclopropyl )-9-fluoro-3-methyl-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid. HPLC confirmation validated the reliability of the proposed FPIA for use as an ideal rapid-screening and high-throughput tool. This is the first FPIA with a structurally heterologous tracer to improve the sensitivity for the detection of clinafloxacin present in goat milk.
MATERIALS AND METHODS Reagents and instrumentation Fluorescein isothiocyanate isomer I (FITC) and triethylamine were purchased from Sigma (St. Louis, MO, USA). Anti-clinafloxacin (anti-CLI) polyclonal antibody was generated previously by Guangdong Provincial Key Laboratory of Food Quality and Safety.
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Silica gel 60 aluminum sheets (type GF254, layer thickness 0.25 mm) for thin-layer chromatography (TLC) were purchased from Merck (Darmstadt, Germany). The analytical standards (clinafloxacin, pazufloxacin (PAZ), pipemidic acid (PIP), enoxacin (ENO), norfloxacin (NOR), ciprofloxacin (CIP)) were donated by the veterinary college of South China Agricultural University (Guangdong, China). Goat milk was obtained from a local farm (Moscow, Russia) and was assumed as initially FQ free. Other reagents and organic solvents were analytical grade. Borate buffer (BB, 0.05 mol/L, pH 8.0) with 0.1% Tween-20 and 0.1% methanol (BBTM) was used as the working buffer for all FPIA experiments. Phosphate buffer (PBS, 0.1 mol/L, pH 7.4) with 2% methanol (PBSM) was used as the working buffer for aqueous standard solutions of the analytes. FPIA was conducted using a TDX/FLX Analyzer (Abbott Laboratories, Irving, TX USA) in semi-automatic PhotoCheck mode. TDX/FLX glass cuvettes (up to 10 in one run) were loaded into the special carousel, followed by the measurement of polarization (in mP units) and the intensity (in conventional units) of fluorescence.
Synthesis of fluorescein-labeled tracers The FITC-labeled pazufloxacin (tracer PAZ-FITC) was synthesized as below. Briefly, 2.0 mg pazufloxacin was dissolved in methanol (1.0 mL). Then, 160 μL FITC solution (10 mg FITC was dissolved in 0.8 mL methanol) and 0.2 mL triethylamine were added dropwise to the solution. After stirring at room temperature overnight in the dark16, small portions of the reaction mixture were first separated by TLC using CHCl3: methanol= 1:1(V:V) as the
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eluent. The plates were dried and redeveloped with CHCl3: methanol: ammonia= 5:2:0.4 (V: V: V). The major yellow bands of varying Rf values were collected, eluted with 0.5 mL methanol and stored at −20℃ under the conditions of protection from light. The tracer concentration was evaluated as in the previously reported method17. The working tracer concentration was chosen as the fluorescence intensity for free tracer which have fluorescence intensity 10 times higher than buffer at approximately 2000-2500 units. The other five fluorescein-labeled tracers (CLI-FITC, PIP-FITC, ENO-FITC, NOR-FITC, CIP-FITC) were synthesized as in the procedure above (Figure 1). Subsequently, to obtain the optimal tracer with sufficient binding capability to the anti-CLI, each tracer was diluted and mixed with a fixed antibody diluted solution. A series of calibration curves was constructed and the binding activity evaluation was completed by comparing three parameters (δmP, IC50 and δmP/IC50). Herein, δmP means the difference between the maximal and minimal fluorescence polarization signals. Lower IC50 values and higher values of δmP and δmP/IC50 indicate a more sensitive assay18.
Fluorescence polarization immunoassay procedure 50 μL clinafloxacin standard solution was mixed with 500 μL tracer-diluted solution, and 500 μL of the diluted antibody was used to construct a series of calibration curves for the selection of the optimal antibody dilution according to three parameters (δmP, IC50 and δmP/IC50) 18. The clinafloxacin standard solution (50 μL), at concentrations from 0.1 μg/L to 100,000 μg/L (0.1 g/L), was mixed with the diluted tracer (500 μL) and the diluted antibody (500 μL)
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to construct the competitive calibration curves. The mP values were plotted against the analyte concentration, and a four-parameter equation was used to fit the experimental sigmoidal curve using Origin 8.5 for Windows19-21: y= (A-D)/ [1+(x/C)B]+D In the equation, A is the maximal mP, D is the minimal mP, C is the clinafloxacin concentration at 50% of tracer binding (IC50) and B is the slope of the curve. The working range represents the standard concentration at 20%–80% of tracer binding (IC20－IC80)22, the limit of detection (LOD) was defined as the standard concentration at 10% of tracer binding22, and the limit of quantification (LOQ) was set as the standard concentration of 20 % tracer binding23-25.
Specificity The specificity of the FPIA was determined using 6 kinds of various FQs under optimized FPIA conditions. Cross-reactivity (CR) was calculated according to the following equation, where IC50 is the concentration at which 50% of the anti-CLI is bound to the analyte. CR%= [IC50 (clinafloxacin)/IC50 (structurally related compounds)] ×100%
Recovery To evaluate the recovery of the developed FPIA, goat milk was spiked with clinafloxacin and the recoveries were determined by FPIA. Three concentrations of the clinafloxacin standard were added to the goat milk samples. Saturated ammonium sulfate
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solution was added to each goat milk solution (V: V=1:1) to precipitate protein. Then, the samples were centrifuged for 10 min (13,000 r/min, 4 °C) to remove the fat from the goat milk. The lower liquid nonfat phase was diluted 1:4 in borate buffer before being used in FPIA. Blank samples were prepared as described above but were not spiked with analyte. Finally, the clinafloxacin concentration in spiked samples was detected by the established FPIA.
HPLC confirmation Goat milk samples for HPLC analysis were prepared according to the method described by Chinese National Standard (GB 29692-2013) 26. Briefly, homogenized goat milk samples (2 mL), spiked with different concentrations of clinafloxacin, were added to 100 μL phosphoric acid and 4 mL acetonitrile and stirred for 5 min on a shaker. After centrifugation (10,000×g, 10 min), the supernatant was transferred into a centrifuge tube. Then, 5 mL n-hexane was added to the supernatant and stirred for 1 min. After several minutes of standing, the subnatant was transferred into a 25 mL specimen bottle. The residue was again extracted with 4 mL acetonitrile. Both extraction solutions were mixed and evaporated to dryness at 50 ℃. The residue was redissolved in 1 mL mobile phase (0.05 mol/L phosphoric acid-triethylamine (pH 2.4) and acetonitrile (90:10, v/v)), and the solution was filtered (0.22 μm) and transferred to an HPLC sample vial ready for analysis. HPLC analysis was performed by a Waters E2695 HPLC with a fluorometric detector (model 2475). The excitation wavelength and emission wavelength were 290 nm and 490 nm, respectively. A C18 column (150 × 4.6 mm, 5 μm particle size; Agilent) was used as the
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stationary phase. The flow rate was 1 mL/min, and the column temperature was 30℃. The measured results were compared with the FPIA results. The linear correlation between the results of two methods was verified by the Student's t-test.
RESULTS AND DISCUSSION Effect of tracer structure on FPIA sensitivity The tracer structure could greatly influence the immunoassay performance
27
. Among
the obtained six tracers (the homologous one (CLI-FITC) and the heterologous ones (PAZ-FITC, PIP-FITC, CIP-FITC, ENO-FITC and NOR-FITC)), compared with the negative one, each tracer showed δmP value from 90 to 199 mP, which could demonstrate sufficient binding capability with the employed antibody against clinafloxacin (Figure 2)28-30. Thus, the working concentration of these tracers in FPIA was approximately 4 nmol/L, as in the previously reported method17. According to the antibody dilution curve, the concentration demonstrating 70% binding with the antibody was selected as the working concentration31, 32. Herein, the 1/200 dilution titer was selected as the working concentration of antibody, and thus, the calibration curve for clinafloxacin with different tracers was plotted (Table 1). Compared to the homologous tracer CLI-FITC, all four of the heterologous tracers (PAZ-FITC, PIP-FITC, ENO-FITC and NOR-FITC) demonstrated a significantly lower IC50 (2-6 times lower). PAZ-FITC, exhibited the highest δmP (150), the lower IC50 (29.3 μg/L) and the maximal δmP/IC50 (5.12); its IC50 showed it had 6 times higher sensitivity than the homologous tracer CLI-FITC. This could be ascribed that a heterologous tracer usually results in weaker recognition by the antibody than
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the recognition to the analyte. The analyte mimics the immunizing hapten (used for the immunogen) more closely than the tracer hapten33, 34, as a result, the affinity of the analyte to the antibody has likely been relatively improved 35 and the detection sensitivity was enhanced accordingly. The optimized, typed calibration curve for clinafloxacin is shown in Figure 3. The LOD (10% inhibition concentration) was 4.1 μg/L, the LOQ (20% inhibition concentration) was 8.5 μg/L, and the working range (20%–80% inhibition concentration) was 8.5－100.5 μg/L. The performance assay of this proposed FPIA could meet the requirements for the FQ residue limitation in China, Korea and the EU5, 6.
Specificity The cross-reactivities (CRs) of 6 kinds of FQs were determined using the developed FPIA with clinafloxacin as the reference compound (CR=100%) (Table 2). The CRs for pazufloxacin and ciprofloxacin were 26.6% and 32.5%, respectively, while the others were lower. Comparing the structures of clinafloxacin and ciprofloxacin, a primary amine at position 7 for clinafloxacin was changed to a dinonyl amine in ciprofloxacin. The CR decreased from 100% for clinafloxacin to 32.5% for ciprofloxacin, implying that the amine group was likely a key structural factor for antibody recognition. In addition, although enoxacin differs from pipemidic acid in structure only by the group at position 6, the antibody showed a higher recognition to enoxacin (CR, 10.9%) than pipemidic acid (CR, 4.7%), suggesting that position 6 is also an important site for antibody recognition. Pazufloxacin showed a CR of 26.6% to the antibody, however, to date, it was a human
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used antibiotic to treat infections of the respiratory tract, urinary-reproductive tract and post-operative infections36-38, as no country has permitted it to be used in animal reproduction and treatment. Therefore, pazufloxacin will seldom occur in goat milk. Ciprofloxacin is a broad-spectrum FQ, and it was often reported to exhibit broad CR with other FQs due to the shared common structure of other FQ drugs39, 40. It seems that the high CR from ciprofloxacin is not usually evitable in the immunoassay of FQ drugs26, 27 but a statutory confirmatory method is usually still needed to exclude the false positive after a screening assay41. For a rapid screening purpose, the FPIA with low CR is acceptable considering the speed and convenience of an FPIA42, 43.
Recovery The average recovery of clinafloxacin from fortified goat milk was 96.6%, ranging from 86.8%–104.5% (Table 3), and the average relative standard deviation (RSD) was 5.3%, ranging from 4.1%–7.2%. This indicated that the recovery and reproducibility of the developed FPIA were satisfactory for a rapid screening analysis.
HPLC confirmation The analytical results obtained with the FPIA and HPLC methods showed good correlation (R2 = 0.9665) (Figure 4). The P value of less than 0.01 by Student's t-test was considered significant positive correlation between the results of two methods.
This
suggested that the developed heterologous strategy-based FPIA method could be used as a rapid screening and high-throughput method for monitoring clinafloxacin residue in goat
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milk samples.
CONCLUSIONS Six tracers with different structures were designed to optimize the assay performance. The heterologous structure strategy significantly enhanced the sensitivity up to 6 times compared to the usual homologous assay format. The established FPIA was successfully used for milk samples with excellent specificity, recovery and relative standard deviation. HPLC confirmation also validated the reliability of the developed FPIA. In conclusion, the proposed FPIA could be considered a highly sensitive and rapid high-throughput method for monitoring clinafloxacin residue in goat milk.
ACKNOWLEDGEMENTS This work was supported by Guangdong Planed Program in Science and Technology (cgzhzd0808, 2013B051000072, 2012B090600005), the PhD Programs Foundation of Ministry of Education of China (20114404130002), Guangdong Natural Science Foundation (S2013030013338), Natural Science Foundation of China (U1301214), the Russian State Targeted Program ‘Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014–2020’ (contract 14.613.21.0028, unique identifier of applied research: RFMEFI61314X0028) and the Russian Foundation for Basic Research grants 14-03-00753, 13-03-93000 and 12-03-92105.
REFERENCES
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1 Sweeney MT, Quesnell R, Tiwari R, LeMay M and Watts JL, In vitro activity and rodent efficacy of clinafloxacin for bovine and swine respiratory disease. Front Microbiol 4:154 (2013). 2 García-Rodríguez JA, Sanchez JG, Trujillano I, Sánchez EG, Garcia MG and Fresnadillo M, Susceptibilities of Brucella melitensis isolates to clinafloxacin and four other new fluoroquinolones. Antimicrob Agents Ch 39:1194-1195 (1995). 3 Pirisi A, Lauret A, Dubeuf JP. Basic and incentive payments for goat and sheep milk in relation to quality. Small Ruminant Res 68:167-178(2007). 4
Liu YZ, Jiang LQ and Xi HP, Research progress of determination method for fluoroquinolones residue in milk and dairy products. Food Ind 34:185-188 (2013).
5 Ministry of Agriculture of the People’s Republic of China, Maximum residue limits for veterinary drugs in animal products. (2002). 6 Korea Food and Drug Administration, Korea residue limits of veterinary drugs in food. (2008). 7 Official Journal of the European Communities. Commision Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. (2009). 8 Zhou MH, Yu YQ, Duan GL, Cheng WB, Liu X, Cao Q and Zhang Y, Determination of clinafloxacin in rat plasma by HPLC. Chin Pharm Anal 21:270-273 (2001). 9 Navalón A, Araujo L, Prieto A and Vı́lchez JL, Determination of grepafloxacin and clinafloxacin by capillary zone electrophoresis. J Chromatogr B 772:65-72 (2002). 10 Xu Q, Zhang JX, Shi NN and Zhou XX, Determination of clinafloxacin in plasma of rats
This article is protected by copyright. All rights reserved
by RP-HPLC. Chin J Pharm 31:316-318 (2000). 11 Wang WS, Shih CW, Hung SW, Ling YF, Tu CY, Chen BR, Tsou LT and Ho SP, Development and application of the enzyme-linked immunosorbent assay residual detection kits for fluoroquinolones. Taiwan Vet J 32:391-403 (2006). 12 Wen K, Nolke G, Schillberg S, Wang ZH, Zhang SX, Wu CM, Jiang, HY, Meng H and Shen JZ, Improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20 fluoroquinolones. Anal Bioanal Chem 403:2771-2783 (2012). 13 Kuhlhoff S and Diehl Y, Rapid screening test for determination of fluoroquinolones in commercial poultry meat. Dtsch Lebensm-Rundsch 101:384-388 (2005). 14 Eremin SA. Fluorescence polarisation immunoassays for determination of pesticides and biologically active compounds in food safety and environmental monitoring. Food Technol Biotech 36:235-244(1998). 15 Eremin SA. Polarization fluoroimmunoassay for rapid, specific detection of pesticides. Chapter 16 In: "ACS Symposium Series No.586, Immunoanalysis of Agrochemicals: Emerging Technologies" (Eds. Judd O. Nelson, Alexander E. Karu, and Rosie Wong). American Chemical Society, Washington, DC 223-234 (1995). 16 Yakovleva JN, Lobanova AY, Shutaleva EA, Kourkina MA, Mart’ianov AA, Zherdev AV, Dzantiev BB and Eremin SA, Express detection of nonylphenol in water samples by fluorescence polarization immunoassay. Anal Bioanal Chem 378:634-641 (2004). 17 Hatzidakis GI, Tsatsakis AM, Krambovitis EK, Spyros A, Eremin SA. Use of L-lysine fluorescence derivatives as tracers to enhance the performance of polarization
This article is protected by copyright. All rights reserved
fluoroimmunoassays. A study using two herbicides as model antigens. Anal Chem 74:2513-2521(2002). 18 Lei HT, Xue G, Yu CF, Haughey SA, Eremin SA, Sun YM, Wang ZH, Xu ZL, Wang H and Shen YD, Fluorescence polarization as a tool for the detection of a widely used herbicide, butachlor, in polluted waters. Anal Methods 3:2334-2340 (2011). 19
Oberleitner L, Grandke J, Mallwitz F, Resch-Genger U, Garbe LA, Schneider RJ. Fluorescence Polarization Immunoassays for the Quantification of Caffeine in Beverages. J Agr Food Chem 62:2337-2343(2014).
20
Eremin SA, Ryabova IA, Yakovleva JN, Yazynina EV, Zherdev AV, Dzantiev BB. Development of a rapid, specific fluorescence polarization immunoassay for the herbicide chlorsulfuron. Anal Chim Acta.468:229-236(2002).
21
Benito-Peña E, Moreno-Bondi MC, Orellana G, Maquieira Á, van Amerongen A. Development of a novel and automated fluorescent immunoassay for the analysis of β-lactam antibiotics. J Agr Food Chem 53:6635-6642(2005).
22 Murtazina NR, Eremin SA, Mozoleva OV, Everest SJ, Jim Brown A and Jackman R, Fluorescent polarization immunoassay for sulphadiazine using a high specificity antibody. Int J Food Sci Tech 39:879-889 (2004). 23
Parra J, Mercader JV, Agulló C, Abad-Somovilla A, Abad-Fuentes A. Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays. Anal Chim Acta 715:105-112(2012).
24
Suarez-Pantaleon C, Wichers J, Abad-Somovilla A, van Amerongen A, Abad-Fuentes
This article is protected by copyright. All rights reserved
A. Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron. Biosens Bioelectron 42:170-176(2013). 25
Garcia-Fernandez J, Trapiella-Alfonso L, Costa-Fernandez JM, Pereiro R, Sanz-Medel A. A Quantum Dot-Based Immunoassay for Screening of Tetracyclines in Bovine Muscle. J Agric Food Chem 62:1733-1740(2014).
26 Ministry of Agriculture of the People’s Republic of China, National Health and Family Planning Commission of the People’s Republic of China, Determination of quinolones residues in milk by high performance liquid chromatographic method (GB 29692-2013). (2013). 27 Choi EH, Kim DM, Choi SW, Eremin SA and Chun HS, Optimisation and validation of a fluorescence polarisation immunoassay for rapid detection of zearalenone in corn. Int J Food Sci Tech 46:2173-2181 (2011). 28
Wang ZH, Cheng LL, Shi WM, Zhang SX, Shen JZ. Fluorescence polarization immunoassay for salinomycin based on monoclonal antibodies. Sci China Chem 53:553-555(2010).
29 Mi TJ, Wang ZH, Eremin SA, Shen JZ, Zhang SX. Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay. J Agric Food Chem 61:9347-9355(2013). 30
Zhang J, Wang ZH, Mi TJ, Wenren LQ, Wen K. A homogeneous fluorescence polarization immunoassay for the determination of cephalexin and cefadroxil in milk. Food Anal Methods 7:879-86(2014).
This article is protected by copyright. All rights reserved
31
Ren LL, Meng M, Wang P, Xu ZH, Eremin SA, Zhao JH, Yin YM, Xi RM. Determination of sodium benzoate in food products by fluorescence polarization immunoassay. Talanta.121:136-143(2014).
32
Mi TJ, Wang ZH, Eremin SA, Shen JZ, Zhang SX. Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay. J Agric Food Chem. 61:9347-9355(2013).
33
Wang ZH, Zhang SX, Shen JZ and Eremin SA. Analysis of sulfamethazine by fluorescence polarization immunoassay. Chin J Anal Chem 35:819-824(2007).
34
Xu ZL, Wang Q, Lei HT, Eremin SA, Shen YD, Wang H, Beier RC, Yang JY, Maksimova KA and Sun YM. A simple, rapid and high-throughput fluorescence polarization immunoassay for simultaneous detection of organophosphorus pesticides in vegetable and environmental water samples. Anal Chim Acta 708:123-129(2011).
35 Zhang Q, Wang LB, Ahn KC, Sun Q, Hu BS, Wang J and Liu FQ, Hapten heterology for a specific and sensitive indirect enzyme-linked immunosorbent assay for organophosphorus insecticide fenthion. Anal Chim Acta 596:303-311(2007). 36
Nomura T, Hirai K, Yamasaki M, Inoue T, Takahashi M, Kawashima T, Sato F and Mimata H. Efficacy of prophylactic single-dose therapy using fluoroquinolone for prostate brachytherapy. Jpn J Radiol 30:317-322(2012).
37 Luo YL, Mo BW, Yin YS, Zhang ZB, Yan SF, Yue HM and Cao XL. A multiple center randomized controlled clinical study of pazufloxacin mesylate for injection in treatment of patients with acute bacterial infections. Chin J New Drugs Clin Rem 32:534-538(2013).
This article is protected by copyright. All rights reserved
38 Uegami S, Ikawa K, Ohge H, Nakashima A, Shigemoto N, Morikawa N, Murakami Y, Sueda T, Uegami S, Ohge H, Nakashima A, Shigemoto N, Murakami Y and Sueda T. Pharmacokinetics
and
pharmacodynamic
target
attainment
of
intravenous
pazufloxacin in the bile of patients undergoing biliary pancreatic surgery. J Chemother 26:287-292(2014). 39 Duan JH, Yuan ZH. Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues. J Agric Food Chem 49:1087-1089(2001). 40 Jiang JQ, Zhang HT, An ZX, Xu ZY, Yang XF, Huang HG and Wang ZL. Development of an heterologous immunoassay for ciprofloxacin residue in milk. Phys Procedia 25:1829-1836(2012).
41 Official Journal of the European Communities. Commission Decision of 12 August 2002 Implementing the Council Directive 96/23/EC Concerning the Performance of Analytical Methods and the Interpretation of Results (2002/657/EC). (2002). 42 Shim WB, Yakovleva ME, Kim KY, Nam BR, Vylegzhanina ES, Komarov AA, Eremin SA and Chung DH. Development of Fluorescence Polarization Immunoassay for the Rapid Detection of 6-Chloronicotinic Acid: Main Metabolite of Neonicotinoid Insecticides. J Agric Food Chem 57:791-796(2009). 43 Li M, Liu XF, Hua XD, Yin W, Fang QK and Wang MH. Fluorescence polarization immunoassay for highly efficient detection of clothianidin in agricultural samples. Anal Methods 6:6541-6547(2014).
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Figure captions
Figure 1 Schematic routes for the synthesis of 6 kinds of (fluoro)quinolone tracers. Figure 2 Dilution curves of the anti-clinafloxacin antibody. Figure 3 FPIA calibration curve for clinafloxacin (n=3). Figure 4 Correlation between FPIA and HPLC for spiked goat milk (n=3).
Figure 1 Schematic routes for the synthesis of 6 kinds of (fluoro)quinolone tracers.
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PAZ-FITC CLI-FITC PIP-FITC ENR-FITC NOR-FITC CIP-FITC Negative
mP
300
200
100
0
100
1000
10000
Dilution curve of antibody
Figure 2 Dilution curves of the anti-clinafloxacin antibody.
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Table 1 Analytical characteristics of FPIA with homologous and heterologous tracers. Antibody
anti-CLI
Titer of antibody
Tracer
δmP
1/200
PAZ-FITC CLI-FITC PIP-FITC ENO-FITC NOR-FITC CIP-FITC
150 120 55 40 45 55
IC50 (μg/L) 29.3 181.0 31.3 37.6 27.3 113.1
δmP/IC50 5.12 0.66 1.76 1.06 1.65 0.49
IC20－IC80 (μg/L) 8.5－100.5 39.0－837.4 8.1－120.5 8.1－174.4 7.7－96.7 32.7－391.3
LOD (μg/L) 4.1 15.9 3.7 3.3 3.7 15.8
δmP means the response span between the maximal and minimal fluorescence polarization signals 18.
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Table 2 Cross-reactivity (CR) for various fluoroquinolones with clinafloxacin No 1 2 3 4 5 6
Compound Clinafloxacin Pazufloxacin Pipemidic acid Enoxacin Norfloxacin Ciprofloxacin
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CR IC50 (nmol/mL) (%) 0.07 100.0 0.27 26.6 1.54 4.7 0.60 10.9 0.67 10.9 0.22 32.5
Table 3 Recovery of clinafloxacin from spiked goat milk samples detected by FPIA (n=3) Spiked level (μg/L) 100 250 500
Found level (μg/L)
Recovery (%, n=3)
99.0±4.0 249.1±11.8 455.9±32.8
99.0±4.0 99.6±4.7 91.2±6.6
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Mean recovery (%)
RSD (%)
Mean RSD (%)
96.6
4.1 4.8 7.2
5.3
mP
220
PAZ-FITC, 4 nmol/L anti-CLI, 1/200 IC50=29.3 μg/L
200 180
LOD=4.1 μg/L Working range=8.5−100.5 μg/L Equation: y=151.5 / [1+x/29.3]1.1+53.9 R2=0.99985
160 140 120
O
100 80
HCl H2 N
O
F
OH
N
N Cl
60 40 10-2 10-1
100
101
102
103
104
105
106
Concentration of clinafloxacin,μg/L
Figure 3 FPIA calibration curve for clinafloxacin (n=3).
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HPLC (μg/L)
400
300
200
y=0.87x-13.31 R2=0.9665
100
100
200
300
400
500
FPIA (μg/L)
Figure 4 Correlation between FPIA and HPLC for spiked goat milk (n=3).
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