Simultaneous Determination of 13 Fluoroquinolone and 22

Letter pubs.acs.org/ac

Simultaneous Determination of 13 Fluoroquinolone and 22 Sulfonamide Residues in Milk by a Dual-Colorimetric Enzyme-Linked Immunosorbent Assay Wenxiao Jiang,† Zhanhui Wang,† Ross C. Beier,‡ Haiyang Jiang,† Yongning Wu,§ and Jianzhong Shen*,† †

Department of Pharmacology and Toxicology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845-4988, United States § China National Center for Food Safety Risk Assessment, Beijing 100021, China ‡

S Supporting Information *

ABSTRACT: Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor two classes of antimicrobial residues in different food matrixes. In this paper, we describe a dual-colorimetric ELISA for the simultaneous detection of 13 fluoroquinolone and 22 sulfonamide residues. The limit of detection for fluoroquinolones and sulfonamides was 2.4 and 5.8 ng/mL, respectively. The developed immunoassay is suitable for high-throughput screening of these low-molecular weight contaminants. This is the first report where two different enzymes (alkaline phosphatase and horseradish peroxidase) were used in one immunoassay and together in a single well for simultaneous detection of multiple low-molecular weight chemical residues.

F

Immunoassays have been confirmed as an alternative analysis method to chromatography methods and are useful for screening purposes by virtue of their high sample throughput, sensitivity, and selectivity, as well as reliability and simplicity.13 New immunoassays are primarily aimed at (a) increasing specificity and sensitivity, (b) reducing assay time and simplifying the procedure, and (c) determining several analytes simultaneously (see Trends in Residue Analysis by Immunoassay in the Supporting Information).14 In this paper, a novel dual-colorimetric enzyme-linked immunosorbent assay (DCELISA) was developed for the simultaneous screening of 13 FQ and 22 SA residues in milk. This is the first report where two different enzymes, alkaline phosphatase (ALP) and horseradish peroxidase (HRP), were used at the same time in each well of a single immunoassay for simultaneous detection of multiple lowmolecular weight chemical residues from two different chemical classes.

luoroquinolones (FQs) and sulfonamides (SAs) are the most commonly prescribed antimicrobial agents in animal husbandry for treating infections, as well as for growth promotion.1,2 Both FQs and SAs are broad-spectrum synthetic antibiotics used against most Gram-positive and many Gramnegative microorganisms and protozoa.3,4 Widespread use of FQs and SAs in animal husbandry without using the proper withdrawal times could lead to the accumulation of chemical residues in eggs, meat, and milk, as well as fish. The presence of these chemical residues in food constitutes a potential hazard for humans due to allergic or toxic reactions.5 Furthermore, the extensive use of FQs and SAs in animal husbandry have been associated with the increasing prevalence of multidrug resistant pathogens, which has caused worldwide concern.6 Many countries have established regulatory rules for using these antimicrobial agents in food animals in order to protect consumers from exposure to harmful residues. The European Union has set maximum residue limits (MRLs) for several FQs ranging from 10 μg/kg for sarafloxacin in chicken fat to 1 900 μg/kg for difloxacin in poultry liver.7 The MRLs for sulfonamides have been set to 100 μg/kg in the United States, the European Union, and China.8,9 During the last two decades, various chromatography methods have been developed for the determination of FQs and SAs in different food matrixes.10,11 Monitoring these chemical residues in real-time requires technology for precise, on-site detection without the need for complicated sample preparation, expensive equipment, and trained personnel.12 © 2013 American Chemical Society

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EXPERIMENTAL SECTION

Synthesis of Antigens and Production of Antibodies. In this work, we covalently attached BSA to 4-(4-(4aminophenylsulfonamido)phenyl)butanoic acid, which provided a new SA immunogen (see Supplemental Figure 1 in the Supporting Information). The hapten sulfathiazole (ST) Received: December 12, 2012 Accepted: January 24, 2013 Published: January 24, 2013 1995

dx.doi.org/10.1021/ac303606h | Anal. Chem. 2013, 85, 1995−1999

Analytical Chemistry

Letter

Figure 1. Schematic representation and standard curves of the DC-ELISA.

buffer. The half maximal inhibitory concentration (IC50) represents the concentration of an inhibitor that is required for 50% inhibition of antibody binding to the coating antigen (B/B0 = 50%). Competition curves were graphed by plotting the OD values against the log10 of the analyte concentration, and the OD values were fitted using a four parameter logistic equation in order to calculate the IC50 value.16 The specificity of the DC-ELISA was evaluated by determining the cross-reactivity with a set of structurally related analytes. The cross-reactivity values were calculated according to the following equation: cross-reactivity (%) = [IC50 (CIP/SM2)/IC50 (analyte)] × 100.17 Sample Preparation and Assay Validation. Commercial milk samples were purchased from local supermarkets in Beijing, China. All samples were determined to be free of FQs and SAs before use by analyzing them with a previously published instrumental method.18 Milk samples were analyzed with the DC-ELISA format using the following protocol: Briefly, the milk samples were defatted by centrifugation at 5000g for 10 min at 4 °C. Then, 5 mL of the defatted milk samples were transferred into individual 50 mL polypropylene centrifuge tubes. Following the addition of 0.2 mL sodium nitroprusside (0.36 M) and 0.2 mL zinc sulfate (1.04 M), the samples were vortexed for 1 min and then centrifuged at 3000g for 10 min at 10 °C. The supernatant was removed and diluted 10 times with PBS for determination by the developed DCELISA. The limit of detection (LOD) was defined as the concentration corresponding to the mean value of 20 blank samples, plus three times the standard deviation. The recoveries of 5 FQs and 5 SAs in milk were determined to evaluate the accuracy and precision of the developed DC-ELISA. Contaminated blind milk samples were prepared at the Veterinary Drug Safety Inspection and Testing Center of the Ministry of Agriculture (Beijing, China) by spiking fresh milk samples with the appropriate standards. Prior to sample extraction, as described above, each sample was spiked at three concentration levels (a single sulfonamide and a single fluoroquinolone together) and run in triplicates. The accuracy of the DC-ELISA was studied by comparing the spiked sample concentration to their measured values by the following equation: recovery (%) = [concentration measured/concentration spiked] × 100. The precision of the DC-ELISA method was assessed on the basis of the relative standard deviation (RSD). The interassay

was covalently linked to OVA for use as the coating antigen (ST-OVA) by the diazotization reaction (see Supplemental Figure 1 in the Supporting Information). The hapten norfloxacin was covalently linked to BSA for use as an immunogen and OVA for use as the coating antigen (NOROVA) using the N-hydroxysuccinimide ester method (see Supplemental Figure 2 in the Supporting Information). Procedures for generation of monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs) against hapten−BSA conjugates (i.e., immunization, cell fusion, hybridoma selection, and cloning) were similar to those previously described.15 The antibody development work was carried out by Beijing WDWK Bio Co. (Beijing, China). For more experimental section details, see Supplemental Experimental Information in the Supporting Information. Development of the DC-ELISA. Before development of the DC-ELISA, individual ELISAs for FQs and SAs had to be established. The best working concentration of the immunoreagents (coating antigen and antibody) was determined using a checkerboard titration. The DC-ELISA approach can be described as follows: microtiter plates were coated with NOR-OVA and ST-OVA (100 μL/well), and then, the plates were incubated at 4 °C overnight. After blocking, 50 μL of diluted ciprofloxacin (CIP) and sulfamethazine (SM2) standard solutions and 50 μL of the diluted anti-FQ mAb and anti-SA PAb were added to each well, and the plates were incubated for 30 min at 37 °C. After washing, 100 μL of diluted goat-antimouse IgG-ALP and goatantirabbit IgG-HRP solutions were added to each well, and the plates were incubated for 30 min at 37 °C. After washing, the ALP substrate solution (100 μL) was added and the optical density (OD) was determined at 405 nm after incubation at 37 °C for 10 min. After discarding the ALP substrate solution, the plates were washed four times and then the HRP substrate solution (100 μL) was added. After incubation at 37 °C for 15 min, the colorimetric reaction was stopped by adding 50 μL of 2 M H2SO4 to each well and the OD values were then determined at 450 nm. The schematic representation of the DC-ELISA is shown in Figure 1. Sensitivity and Specificity Determination. Sensitivity and specificity are important parameters for the evaluation of ELISA methods. The OD value of the wells containing only assay buffer was referred to as B0. The OD values of the standards were normalized against the OD value of the assay 1996



Letter

Table 1. IC50 and Cross-Reactivity Using Ciprofloxacin and Sulfamethazine as Standard Curves fluoroquinolones

sulfonamides

analytes

CR (%)

analytes

CR (%)

analytes

CR (%)

ciprofloxacin (standard curve) enrofloxaxin norfloxaxin ofloxacin marbofloxacin danofloxacin lomefloxacin pefloxacin orbifloxacin enoxacin sparfloxacin oxilinic acid flumequine

100 90 116 58 43 129 130 93 64 150 15 409 132

sulfamethazine (standard curve) sulfamonomethoxine sulfisomidine sulfaquinoxaline sulfadimoxine sulfachloropyridazine sulfamethoxydiazine sulfapyridine sulfadimethoxine sulfamethoxypyridazine sulfadimoxine sulfacetamide

100 3930 1222 1048 956 740 516 470 440 298 285 220

sulfamerazine sulfisoxazole sulfanitran sulfamethoxazole sulphadiazine sulfathiazole sulfamoxol sulfamethizole sulfabenzamide phthalylsulfathiazole

220 169 147 100 92 85 52 30 14 9

carried out without the addition of a stop solution. Not using a stop solution with the ALP-mediated colorimetric reaction helped to retain the enzyme activity of HRP, which was a key element in improving the HRP colorimetric reaction. Sensitivity and Specificity Determination. The IC50 values were obtained from the standard curves, which were used to evaluate the sensitivity of the developed DC-ELISA. The standard curve was based on a solution containing both CIP and SM2 as the reference calibration standard in the developed DC-ELISA as shown in Figure 1. The IC50 values were 1.4 and 6.2 μg/L for CIP and SM2, respectively. The FQ CIP was used as the standard in the DC-ELISA for determination of FQ residues, and SM2 was used as the standard for determination of SA residues in milk. As shown in Table 1, the anti-FQ mAb exhibited high cross-reactivity against 13 FQs and showed negligible cross-reactivity with others. The anti-SA PAb exhibited high cross-reactivity with 22 SAs. The cross-reactivities are summarized in Table 1. Therefore, on the basis of the broad FQ-specific mAb and broad SA-specific PAb, a DC-ELISA was developed for determination of 13 FQs and 22 SAs in milk. Sample Preparation and Assay Validation. The matrix effect is a common challenge when developing an immunoassay for veterinary drug residue analysis. The complicated food matrix can cause false positives by lowering the intensity of color development or by interfering with the antigen−antibody binding. These types of matrix effects can often be reduced by good sample preparation, such as by a dilution of the samples or by using special cleanup procedures.19 To evaluate whether the milk matrix was effecting the DC-ELISA, standard curves were generated in PBS and compared with standard curves generated using control milk. The effects of dilution were investigated by using a 10- and 20-fold dilution of the milk samples. Dilution of the milk samples did reduce the interfering milk matrix to insignificant levels for the FQs but not for the SAs. The effects of adding BSA, OVA, and Tween 20 to PBS extracts were examined, and there was no obvious improvement with these additions. After removal of the milk protein, the milk sample supernatants were diluted 1:10 with PBS, and this procedure did reduce the matrix effects sufficiently for simultaneous detection of both the FQs and SAs. The LODs of the CD-ELISA method were 2.4 μg/kg for 13 FQs and 5.8 μg/kg for 22 SAs in milk, which meet the MRL requirements. During the spike and recovery tests, no positive results were obtained for the nonspiked samples. Raw milk

repeatability was determined by repeated analysis (n = 4) of blank samples spiked with three different concentrations of analytes.

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RESULTS AND DISCUSSION Development of the DC-ELISA. To establish a sensitive ELISA method, the lowest IC50 value with an adequate OD value (ranging from 1.5 to 2.0) is required. Therefore, the best working concentration of the coating antigen NOR-OVA and corresponding mAb was determined to be 0.2 and 0.7 μg/mL, respectively. The concentration of goat-antimouse IgG-ALP used was the recommended dilution of 1:1 000 (0.06 μg/mL). The optimal working concentration of coating antigen ST-OVA and corresponding PAb was determined to be 0.16 and 0.3 μg/ mL, respectively. Again, the concentration of goat-antirabbit IgG-HRP used was the recommended dilution of 1:5 000 (0.24 μg/mL). During the development of the DC-ELISA, the microplate was coated with a mixture of the FQ coating antigen NOR-OVA and the SA coating antigen ST-OVA. The standard calibration curves were eight-point curves containing both CIP and SM2 analytes. The antibody used was a mixture of the mAb for the FQs and the PAb for SAs, while the enzyme tracer was a mixture of goat-antimouse IgG-ALP and goat-antirabbit IgGHRP. The enzymes most often used in enzyme immunoassays are HRP and ALP. Generally, the ALP enzyme is most stable in alkaline pH, whereas the enzyme HRP is most stable in acidic pH. The ALP substrate was prepared as an alkaline solution, and the HRP substrate was prepared as an acidic solution. Thus, the colorimetric reactions were carried out in a sequential detection process: the ALP colorimetric reaction was carried out first, and then, the HRP colorimetric reaction was carried out (Figure 1). The greatest difficulty facing the DC-ELISA was to provide accuracy for FQ detection without losing sensitivity during detection of the SAs. The colorimetric reactions were carried out sequentially with the ALP enzymatic reaction first followed by the HRP reaction. However, the enzyme activity of HRP was influenced by the reagents used during the ALP colorimetric reaction. The influence could be decreased to insignificant levels by decreasing the time of the ALP colorimetric reaction and by increasing the time of the HRP colorimetric reaction. Theoretically, the colorimetric reaction could be stopped by adding a strong acidic, basic, or chelating agent solution, which will stop the enzyme reaction. In the present study, the ALP-mediated colorimetric reaction was 1997



Letter

Table 2. Mean Recovery and Relative Standard Deviation (RSD) for Five FQs and SAs in Milk (n = 4) FQ analytes

spikeda

recovery (%)

RSD (%)

ciprofloxaxin

10 50 100 10 50 100 10 50 100 10 50 100 10 50 100

87 81 85 72 84 101 91 86 74 92 84 77 91 105 82

5.2 7.7 4.8 9.3 7.7 6.7 5.9 8.2 6.7 6.1 8.3 5.4 7.9 8.3 7.5

enrofloxaxin

norfloxaxin

marbofloxacin

flumequine

a

SA analytes sulfamethazine

sulphadiazine

sulfaquinoxaline

sulfadimethoxine

sulfamethoxydiazine

spikeda

recovery (%)

RSD (%)

10 50 100 10 50 100 10 50 100 10 50 100 10 50 100

76 85 71 67 72 84 95 101 82 72 81 97 80 75 82

9.5 7.9 13.8 11.3 15.6 9.2 12.6 8.1 14.7 8.4 16.4 9.3 7.9 8.8 14.2

The spiked concentrations are in units of μg/kg.

into one immunoassay for the simultaneous determination of 13 FQs and 22 SAs (see Potential Formats of the DC-ELISA in the Supporting Information). The high cross-reactivity for these 35 compounds may affect the detection of other unknown compounds (see Cross-Reactivity in the Supporting Information). The DC-ELISA was performed using the different enzyme labels (ALP and HRP) on the second antibody, and the different analytes can easily be distinguished by the different absorption wavelengths of the enzyme reaction products. In contrast to earlier immunoassays, the DC-ELISA developed here greatly improved the sensitivity and specificity for detection of fluoroquinolones and sulfonamides (see Immunoassay Specificity in the Supporting Information). The DCELISA provides an alternative for a rapid screening procedure for detecting 13 FQs and 22 SAs by immunoassay. The detection limits of this assay meet the requirement of China, the European Union, and the United States.

spiked with FQs and SAs showed good agreement between the spiking level and the concentration detected. As shown in Table 2, the mean recovery for 5 FQs ranged from 72% to 105%, with the RSD values less than 9.3%. The mean recovery for 5 SAs ranged from 67% to 101%, with the RSD values less than 16.4%. As demonstrated using milk samples spiked with 5 FQs and 5 SAs, the novel DC-ELISA method provided satisfactory results for the simultaneous detection of both FQ and SA residues in milk. Comparison with Previous Research. Immunoassays, especially the ELISA, are the most commonly available method for routine screening analysis of various veterinary drug residues in different food matrixes. Over the last two decades, sensitive antibodies have been produced for the determination of FQs in different agricultural products.20−22 In addition, some ELISA methods were also developed for the determination of SAs in dairy products.23−25 These ELISA methods have greatly contributed to the control of FQs and SAs. Traditional immunoassays are performed as discrete tests; that is, one assay for one analyte and several analysis runs are required to determine all of the components in a complex system. Compared with parallel single-analyte immunoassay methods, the multianalyte immunoassay offers some remarkable advantages, such as high sample throughput, improved assay efficiency, low sample consumption, and reduced overall cost per assay.26 These improvements make the multianalyte immunoassay the method of choice for screening samples for multianalytes. A multianalyte immunoassay that can measure two or more chemical classes in one well has become a long established goal.27 The radioimmunoassay using I131 and I125 as labels was reported for the detection of human insulin and growth hormone in serum samples in 1968.28 More recently, dual-label time-resolved fluorescence immunoassays29 have been described for the simultaneous detection of two or more analytes. However, radioactivity poses a potential health hazard, and the preparation of dual label tracers for time-resolved fluoroimmunoassay is complicated and expensive. To the best of our knowledge, there are no reports in the literature that use dual enzyme labels in an ELISA or other immunoassays for the detection of low-molecular weight contaminants. In this study, we combined two enzyme immunoassays, having high crossreactivity for 13 FQs and 22 SAs, and two different enzymes

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CONCLUSIONS In this paper, a novel screening DC-ELISA was developed for the simultaneous determination of 35 low-molecular weight chemical residues (13 FQs and 22 SAs) in milk. It is the first report where the enzyme labels ALP and HRP were used in a single well for simultaneously detecting multiple low-molecular weight chemical residues.

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental experimental information; trends in residue analysis by immunoassay; potential formats of the DCELISA; cross-reactivity; immunoassay specificity; Figure 1, synthesis of immunogen and coating antigen for SAs; Figure 2, synthesis of immunogen and coating antigen for FQs. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-106-273-2803. Fax: +86-106-273-1032. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1998



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Letter

ACKNOWLEDGMENTS This work was supported by the State Key Program of the National Natural Science Foundation of China (Nos. 30830082, 31172631, and 21107104), Special Fund for Agroscientific Research in the Public Interest (No. 201203040), National Science and Technology Pillar Program (Nos. 2012BAK17B16 and 2011BAK10B01-02), and the International Science and Technology Cooperation Program of China (No. 2011DFR30470).

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Simultaneous Determination of 13 Fluoroquinolone and 22

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