Ultrasensitive Immunochromatographic Strip for Fast Screening of 27

Aug 27, 2017 - Collaborative Innovationcenter of Food Safety and Quality Control in Jiangsu ... University, Wuxi, Jiangsu 214122, People's Republic of...
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Ultrasensitive Immunochromatographic Strip for Fast Screening of 27 Sulfonamides in Honey and Pork Liver Samples Based on a Monoclonal Antibody Yanni Chen,†,‡ Lingling Guo,†,‡ Liqiang Liu,†,‡ Shanshan Song,†,‡ Hua Kuang,*,†,‡ and Chuanlai Xu†,‡ †

State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China Collaborative Innovationcenter of Food Safety and Quality Control in Jiangsu Province, Jiangnan University, Wuxi, Jiangsu 214122, People’s Republic of China

‡

ABSTRACT: Group-specific monoclonal antibodies (Mabs) with selectivity for 27 sulfonamides were developed based on new combinations of immunogen and coating antigen. The Mab was able to recognize 27 sulfonamides with 50% inhibition concentration (IC50) values ranging from 0.15 to 15.38 μg/L. In particular, the IC50 values for five sulfonamides (sulfamethazine, sulfaquinoxaline, sulfamonomethoxine, sulfadimethoxine, and sulfamethoxazole) were 0.51, 0.15, 0.56, 0.54, and 2.14 μg/L, respectively. On the basis of the Mab, an immunochromatographic lateral flow strip test was established for rapid screening of sulfonamides in honey samples. The visual limit of detection of the strip test for most sulfonamides in spiked honey samples was below 10 μg/kg, satisfying the requirements of authorities. Positive honey and pork liver samples, which had been confirmed by high-performance liquid chromatography/mass spectrometry, were used to validate the reliability of the proposed strip test. The immunochromatographic lateral flow strip test provides a rapid and convenient method for fast screening of sulfonamides in honey samples. KEYWORDS: monoclonal antibody, sulfonamides, immunochromatographic lateral flow strip test, honey samples, pork liver samples

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INTRODUCTION Sulfonamides are widely used in veterinary medicine for treatment of infectious diseases and as growth-promoting feed additives.1−3 The abuse of sulfonamides in veterinary practice may lead to the presence of sulfonamide residues in foodstuffs derived from animals.4 Excess sulfonamide residues are harmful to consumers because of their carcinogenic potential and the risk of antibiotic resistance.5−7 In order to protect consumers from these risks, the European Union and China have established maximum residue limits (MRLs) for total sulfonamides in edible animal tissues and milk of 100 μg/ kg.8,9 In China, the determination of five sulfonamides (sulfamethazine, sulfaquinoxaline, sulfamonomethoxine, sulfadimethoxine, and sulfamethoxazole) in meat, eggs, and milk is compulsory.8 There are currently more than 20 different sulfonamides used in human and animal healthcare. Figure 1 shows 27 sulfonamides classified according to the chemical group at the N1 position of the common sulfonamide core structure. Chromatography and mass spectrometry are the most commonly used analytical methods for detection of sulfonamides.10−13 Although these methods are sensitive and specific, they require time-consuming pretreatment of samples, highly trained personnel, and expensive instruments. Recently, analytical methods that rely on biosensors have become popular because of their high sensitivity.14−16 In general, enzyme-linked immunosorbent assay (ELISA) and immunochromatographic lateral flow strip test, both based on monoclonal antibody (Mab) are widely used for fast screening in food safety assessment. Compared with the ELISA, the strip test avoids incubation and washing steps and only requires 5− © 2017 American Chemical Society

10 min to fulfill the procedure. Therefore, as a semiquantitative assay, the strip test is undoubtedly more convenient for on-site determination and high-throughput processing of samples. The immunochromatographic strip test for small molecules is based on a competitive format of indirect and competitive ELISA (IcELISA). In brief, a sensitive and broad-specific Mab is essential for establishment of the strip test. There are numerous reports on the generation of groupspecific antibodies for sulfonamides, for both polyclonal antibodies and Mabs. Mabs are favored over polyclonal antibodies because of their high reproducibility and unlimited supply. However, the reported Mabs are generally too specific or recognize only a small group of sulfonamides. For efficient surveillance, an immunoassay that can detect multiple sulfonamides rather than a specific sulfonamide is preferable. In our previous study, a strip test was developed based on a Mab that was able to recognize 26 sulfonamides with IC50 values of 0.08−90.18 μg/L.17 As a consequence of the hapten structure, the Mab showed better inhibition of sulfonamides containing a thiazole ring at the N1 position. The sensitivities of the Mab toward sulfamethazine (18.79 μg/L) and sulfaquinoxaline (39.12 μg/L) were therefore relatively poor. Recent advances in the production of broad-specific Mabs for sulfonamides have been summarized by Wang et al.18 The discussed Mabs recognized eight sulfonamides with IC50 values below 100 μg/L. Moreover, the Mabs (4D11 and 4C7) Received: Revised: Accepted: Published: 8248

July 11, 2017 August 26, 2017 August 27, 2017 August 27, 2017 DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of 27 sulfonamides. The sulfonamides are arranged by the number of rings and the number of atoms in the ring at the N1 position.

Figure 2. Chemical structures of haptens used in this study.

specific Mabs for sulfonamides. In addition, Yuan et al.20 developed a Mab that recognized 16 sulfonamides with IC50 values of 0.52−51 μg/L. Clearly, there is still scope to improve the sensitivity toward some sulfonamides as well as produce

developed by Wang’s group were optimized in their further work.19 Under optimal conditions, the Mab 4D11 showed IC50 values for 22 sulfonamides at concentrations below 100 μg/L, demonstrating significant progress in the production of broad8249

DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

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Journal of Agricultural and Food Chemistry

Figure 3. UV/vis spectra of immunogen and coating antigen. mido) butanoic acid (S4), 2-(4-aminophenylsulfonamido) pyrimidine5-carboxylic acid (S5), 2-(4-aminophenylsulfonamido)-4-methylpyrimidine-5-carboxylic acid (S6), and (E)-5-(2-(4-aminophenylsulfonamido)-4,6-dimethylpyrimidin-5-yl) pent-4-enoic acid (S7). The structures of seven haptens were confirmed by 1H NMR spectra. Hapten S1. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 7.434 (d, 2H, J = 8.8 Hz, CHar), 6.559 (d, 2H, J = 8.4 Hz, CHar), 6.476 (s, 1H, SO2−NH), 5.840 (s, 2H, NH2), 3.469 (s, 2H, CH2−COOH). Hapten S2. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 12.686 (s, 1H, COOH), 10.420 (s, 1H, SO2−NH), 7.792 (d, 2H, J = 8.4 Hz, CHar), 7.468 (d, 2H, J = 8.8 Hz, CHar), 7.165 (d, 2H, J = 8.8 Hz, CHar), 6.560 (d, 2H, J = 8.4 Hz, CHar), 6.050 (s, 2H, NH2) Hapten S3. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 11.944 (s, 1H, COOH), 7.402 (d, 2H, J = 8.8 Hz, CHar), 7.047 (t, 1H, J = 12.4 Hz, SO2−NH), 6.607 (dd, 2H, J = 8.8 Hz, CHar), 5.888 (s, 2H, NH2), 2.639−2.589 (m, 2H, NH−-CH2), 2.163−2.126 (t, 2H, CH2− COOH), 1.431−1.184 (br, m, 6H, CH2−CH2−CH2). Hapten S4. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 12.023 (s, 1H, COOH), 9.714 (s,1H, SO2−NH), 7.367 (d, 2H, J = 8.8 Hz, CHar), 7.023−6.951 (m, 4H, CHar), 6.526 (d, 2H, J = 8.8 Hz, CHar), 5.939 (s, 2H, NH2), 2.476−2.438 (t, 2H, CH2 ph), 2.175−2.139 (t, 2H, CH2COOH), 1.724−1.686 (t, 2H, CH2). Hapten S5. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 8.868 (s, 2H, CHar), 7.652 (d, 2H, J = 8.4 Hz, CHar), 6.075−6.032 (br, 2H, NH2). Hapten S6. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 8.708 (s, 1H, SO2−NH), 7.639 (d, 2H, J = 8.8 Hz, CHar), 6.570 (d, 2H, J = 8.4 Hz, CHar), 2.567−2.505 (d, 2H, CHar). Hapten S7. 1H NMR (400 MHz, DMSO-d6), δ (ppm) 7.640 (d, 2H, J = 8.8 Hz, CHar), 5.945 (s, 2H, NH2), 5.444−5.395 (m, 2H, CHCH), 3.213−2.166 (m, 2H, CH2COOH), 2.876 (d, 2H, J = 5.6 Hz, CH2), 2.328−2.274 (m, 6H, CH3). Haptens S4−S7 were, respectively, conjugated with BSA using EDC/NHS method to obtain immunogens.22,23 All haptens conjugated with OVA using the EDC/NHS method were regarded as coating antigens. Briefly, 17.16 mg of NHS was added to a solution of S4 (24.97 mg dissolved in 3 mL of methanol) with continuous stirring. After 10 min, 28.51 mg of EDC was added to the solution, followed by reaction for 5 h at room temperature. The reaction mixture was then added to a solution of BSA (50 mg dissolved in 5 mL of carbonate buffer (0.05 mol/L, pH 9.6)) and stirred at room

broader specificity Mabs. Therefore, the aim of this study was to develop Mabs with wider applicability, primarily with respect to sensitivity and specificity. Furthermore, an immunochromatographic lateral flow strip test was established based on the proposed Mabs for detection of sulfonamides in honey and pork liver samples.

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MATERIALS AND METHODS

Reagents and Apparatus. Sulfaceamide (1), sulfaguanidine (2), sulfanilamide (3), sulfamethoxazole (4), sulfamethizole (5), sulfathiazole (6), sulfamoxol (7), sulfisoxazole (8), sulfadiazine (9) sulfamerazine (10), sulfamethazine (11), sulfasomidine (12), sulfamonomethoxine (13), sulfameter (14), sulfadimethoxine (15), sulfadoxine (16), sulfamethoxypyridazine (17), sulfachloropyridazine (18), sulfaclozine (19), sulfalene (20), sulfabenzamide (21), sulfapyridine (22), sulfanitran (23), sulfaquinoxaline (24), sulfaphenazole (25), sulfasalazine (26), and phthalylsulfathiazole (27) were purchased from Dr. Ehrenstorfer (Augsburg, Germany) or SigmaAldrich (Shanghai, China). Bovine serum albumin (BSA), ovalbumin (OVA), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (EDC), and N-hydroxysuccinimide (NHS), and enzyme immunoassay-grade horseradish peroxidase labeled goat antimouse immunoglobulin (IgG) were obtained from Sigma-Aldrich. The materials (polyvinyl chloride pads, absorbance pad, sample pad (glass-fiber membrane), and nitrocellulose (NC) membrane) for strip test were obtained from JieYi Biotechnology Co., Ltd. (Shanghai, China). The CM4000 Guillotine Cutting Module and the Dispensing Platform, which were used to obtain individual strips and spray reaction reagents, were purchased from Kinbio Tech Co., Ltd. (Shanghai, China). Absorbance measurements were performed with a spectrophotometric microtiter plate reader (Thermo, Waltham, MA), and UV spectra were determined with an ultraviolet−visible spectrophotometer (Agilent, Santa Clara, CA). Synthesis of Haptens and Immunogens. The haptens were synthesized as previously reported.18,21 The chloride atom of Nacetylsulfaniyl chloride was replaced by amino groups of different chemicals containing carboxyl groups to obtain the following haptens (Figure 2): 2-(2-(4-aminophenylsulfonamido) thiazol-4-yl) acetic acid (S1), 4-(4-aminophenylsulfonamido) benzoic acid (S2), 6-(4-aminophenylsulfonamido) hexanoic acid (S3), 4-(4-(4-aminophenylsulfona8250

DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

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Journal of Agricultural and Food Chemistry

Figure 4. Schematic illustration of the immunochromatographic lateral flow strip test. Immunochromatographic Lateral Flow Strip Test. Preparation of Colloidal Gold. Colloidal gold with a diameter of 20 nm was prepared using the sodium citrate reduction method.23,32,33 Briefly, freshly prepared 1% (w/v) trisodium citrate was added to boiling aqueous HAuCl4·4H2O in a flask with vigorous stirring. The mixture was persistently boiled until the color of the solution changed to winered. The solution was then cooled to room temperature and stored at 4 °C for future use. The colloidal gold was characterized by transmission electron microscopy. Preparation of Colloidal Gold-Mab Conjugates. The procedure for labeling Mab with colloidal gold is well-established in our laboratory.34 In theory, the negatively charged colloidal gold can combine with the positively charged groups of the Mab via electrostatic interaction, which is more stable under weakly alkaline conditions. First, K2CO3 (0.1 M) was used to adjust the colloidal gold to pH 8. The Mab was then slowly added to the colloidal gold solution. To block excess reactivity of colloidal gold, BSA dissolved in ultrapure water was added to the mixture under stirring for 30 min. Centrifugation at 875g for 40 min was fulfilled to remove free blocking agent and excess Mab. The sediment was resuspended twice in borate buffer (0.002 M, pH 8, containing 1% (w/v) sucrose and 0.01% Tween-20). Principle of Strip Test. A competitive format similar to ic-ELISA was the basis of the strip test.35,36 The assembly and principle of the strip is shown in Figure 4. After insertion into the sample extract, the end of the sample pad rapidly wetted. Colloidal gold-Mab immobilized on the conjugate pad is dissolved and begins to flow with the sample up the NC membrane under the capillary effect. The strip is then placed flat to allow the solution to transfer smoothly. Goat antimouse IgG immobilized on the control line can capture the colloidal goldMab, forming a red band that certifies the validity of the strip test. For negative samples, the colloidal gold-Mab can conjugate with both coating antigen on the test line and goat antimouse IgG on the control line, meaning that two red bands appear. In the contrast, for the positive samples, the limited binding site on the colloidal gold-Mab are partially occupied by target analytes in the samples. Therefore, the amount of coating antigen that can combine with colloidal gold-Mab is reduced, resulting in a colorless band on test line than on control line. The higher the concentration of analyte in sample, the less colored the test line is. If the concentration of analyte in the samples is below the LOD, the colors of the two lines cannot be distinguished. The concentration that leads to an obvious difference between test and control lines is defined as the visual limit of detection (vLOD). Analysis of Spiked Honey Samples by Strip Test. The negative honey samples confirmed by HPLC/MS were supported by Jiangsu Entry-Exit Inspection and Quarantine Bureau.37,38 Negative honey

temperature overnight. The solution was then dialyzed to obtain pure immunogen. The other immunogens and coating antigens were prepared similarly. The immunogens and coating antigens were confirmed by their UV spectra as shown in Figure 3. Immunization Schedule. The procedure for immunization was similar to that used in our previous work.24,25 Four immunigens (S4EDC-BSA, S5-EDC-BSA, S6-EDC-BSA, and S7-EDC-BSA, respectively) were used to immunize mice, respectively. After the third immunization, ic-ELISA was performed to screen the sera collected from mice. In the first analysis, seven coating antigens were, respectively, coated on microtiter plates and seven sulfonamides (5, 9, 11, 13, 14, 15, and 24) were applied to screen the mice serum. As a result, the appropriate coating antigen was screened. After the fifth immunization, the mice with high titer and low inhibitory values for the seven compounds were selected for further analysis. This time 27 compounds were used in ic-ELISA to identify the best mouse, and the mouse with broadest cross-reactivity was chosen as the spleen donor. Two days prior to cell fusion, a final intraperitoneal booster injection (25 μg of immunogen directly dissolved in 100 μL of physiological saline) was administered. Cell Fusion and Hybridoma Screening. The cell fusion process was performed as previously described.26,27 Hybridoma screening was conducted with ic-ELISA. After three subclones, the best cell lines were screened and cultured on a large scale. The cell lines were then intraperitoneally injected into mice primed with paraffin to produce ascites. After 7−10 days, ascites were collected and purified (octanoic acid-saturated ammonium sulfate method) to obtain the Mabs. The concentration of Mab was determined by UV/vis spectroscopy at 278 nm. ic-ELISA. The sensitivity and cross-reactivity of the Mabs were evaluated by ic-ELISA. First, the appropriate concentration of coating antigen and Mab were determined using bidimensional titration assay. The detailed procedure was as described in the literature.28,29 The IC50 (concentration of competing compound that produced 50% inhibition of antibody binding to the coating antigen) is considered as an important criterion for evaluation of Mab sensitivity.30,31 The IC50 values of 27 sulfonamides were evaluated, respectively. The ability of structurally related compounds to bind to the Mab is an indicator of specificity.28 Since the IC50 values of 27 sulfonamides were determined, the cross-reactivity (CR) could be calculated according to the following equation:

CR% = (IC50 value of 11)/(IC50 value of related compound) × 100 8251

DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

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Journal of Agricultural and Food Chemistry samples were spiked with different concentrations of 27 sulfonamides, respectively. Honey samples were diluted by phosphate buffered solution (PBS (0.01M, pH 7.2)) two times to eliminate matrix interference. The final concentrations of sulfonamides in honey samples was as follows: 1 (0, 1, 2.5, 5, 10, and 25 μg/kg), 2 (0, 5, 10, 20, and 50 μg/kg), 3 (0, 2.5, 5, 10, and 25 μg/kg), 4 (0, 0.5, 1, 2.5, and 5 μg/kg), 5 (0, 2.5, 5, 10, 25, and 50 μg/kg), 6 (0, 10, 25, 50, and 100 μg/kg), 7 (0, 0.5, 1, 2.5, and 5 μg/kg), 8 (0, 0.5, 1, 2.5, and 5 μg/kg), 9 (0, 0.5, 1, 2.5, and 5 μg/kg), 10 (0, 0.5, 1, 2.5, and 5 μg/kg), 11 (0, 0.25, 0.5, 1, and 2.5 μg/kg), 12 (0, 0.5, 1, 2.5, and 5 μg/kg), 13 (0, 0.5, 1, 2.5, and 5 μg/kg), 14 (0, 0.25, 0.5, 1, and 2.5 μg/kg), 15 (0, 0.5, 1, 2.5, and 5 μg/kg), 16 (0, 0.5, 1, 2.5, and 5 μg/kg), 17 (0, 0.5, 1, 2.5, and 5 μg/kg), 18 (0, 0.5, 1, 2.5, and 5 μg/kg), 19 (0, 0.5, 1, 2.5, and 5 μg/kg), 20 (0, 2.5, 5, and 10 μg/kg), 21 (0, 0.5, 1, 2.5, and 5 μg/kg), 22 (0, 1, 2.5, 5, and 10 μg/kg), 23 (0, 2.5, 5, and 10 μg/kg), 24 (0, 0.1, 0.25, 0.5, 1, and 2.5 μg/kg), 25 (0, 5, 10, 25, and 25 μg/kg), 26 (0, 2.5, 5, 10, and 25 μg/kg), 27 (0, 0.5, 1, 2.5, 5, and 10 μg/kg). Analysis of Positive Samples. The positive honey samples and pork liver sample containing known concentrations of sulfonamides were also provided by Jiangsu Entry-Exit Inspection and Quarantine Bureau. The honey sample (NO.9788) contains 4 at a level of 37 μg/kg and the pork liver sample contains 11 at a level of 110 μg/kg.37,38 On the basis of the spiked-recovery test for 4 and 11, the honey sample was diluted with 5, 10, 20 times, respectively. Therefore, the final concentration of 4 was 7.4, 3.7, and 1.85 μg/kg. Then, the honey samples with different dilution times were analyzed by strip test. A volume of 20 mL of ethyl acetate was added to 5 g of pork liver sample in a 50 mL centrifuge tube and subjected to vigorous shaking for 5 min. The mixture was centrifuged at 875g for 5 min and then the supernatant was collected in a new tube. The sediment was extracted with 10 mL of ethyl acetate, following by centrifugation. The combined supernatants were dried in a stream of nitrogen gas. A volume of 5 mL of 0.01 M PBS was added to dissolve the dried extract. The dissolved extract is then diluted 20, 50, and 100 times with 0.01 M PBS. Therefore, the final concentration of 11 was 5.5, 2.2, and 1.1 μg/ kg. Each of the diluted extract samples were analyzed by a strip test.

be divided into two groups: single-ring haptens and two-ring haptens. The single-ring haptens have been proven to have limited utility for Mab production.17,18,41 In the case of two-ring haptens, most group-specific antibodies showed relatively weak sensitivity against 11, the most important individual sulfonamides. In our previous work,17 Mab generated by hapten S1 exhibited good inhibition for sulfonamides containing a thiazole ring and relatively weak inhibition for sulfonamides with sixmembered ring at N1 position, especially for 11. Mab generated by hapten S2, containing a benzene ring at the N1 position, showed no inhibition for any sulfonamides. We postulated that the two-carbon alkyl chain in the para-position of the benzene ring might be too short, resulting in the benzene ring being hidden by the carrier protein. Therefore, the alkyl chain of hapten S2 was extended to be a four-carbon atom in hapten S4. However, mice sera still showed no inhibition against any sulfonamides. The two-ring haptens containing a benzene ring were rejected based on the results with haptens S2 and S4. We then chose two-ring haptens containing a pyrimidine ring (haptens S5, S6, and S7). Compared with the structure of hapten S5, hapten S6 has an extra side chain and hapten S7 has a longer alkyl chain as well as two side chains. The sera of mice immunized with hapten S5 showed inhibition against most of the sulfonamides using the heterogeneous coating antigen (hapten S3). For hapten S6, we considered that the alkyl chain was too short and that the methyl group on the pyrimidine ring introduced steric hindrance, preventing exposure of the antigenic determinant. The sera of mice immunized with hapten S7 showed good inhibition only for 11. In the case of hapten S7, regardless of the two side chains, the alkyl chain was sufficiently long to expose the antigenic determinant. Furthermore, the pyrimidine ring with two methyl groups was a structural fragment of 11. Therefore, the mice immunized with hapten S7 showed good inhibition for 11. Screening of Antisera. The sera resulting from different haptens were assayed by ic-ELISA with one homogeneous and six heterogeneous coating antigens. The results showed that the combination of hapten S5 (immunogen) and hapten S3 (coating antigen) was optimal. Cross-Reactivity of Mab. The CR of Mab 3D1 was shown in Table 1. As discussed in the section of hapten synthesis, the IC50 values for sulfonamides containing a six-atom pyrimidine ring were low, due to the structure of hapten S5. Moreover, for sulfonamides containing pyrazine or pyridazine rings (isomers of pyrimidine ring), the IC50 values were also good. Recognition of sulfonamides containing a five-membered ring by Mab 3D1 was relatively weak, especially for 6. However, the vLOD of strip test for 6 was still sufficient to satisfy the MRLs of the European Union. The CR data of the reported Mabs 4D11 and 4C718 are also listed in Table 1. Moreover, a comparison of Mab 3D1 and the previously reported Mab 4D11 is shown in Figure 5. It is apparent that the sensitivity and CR of Mab 3D1 were superior to those of Mab 4D11. Analysis of Honey Samples Spiked with Sulfonamides. Strip tests were used to analyze honey samples spiked with sulfonamides. As shown in Figure 6, the vLOD of the three sulfonamides containing a single-ring were 2.5, 10, and 5 μg/kg. With respect to sulfonamides containing a fivemembered ring at the N1 position, the Mab 3D1 has a weaker sensitivity against 6. Therefore, the vLOD of the strip test for 6 was 25 μg/kg, which was higher than for other sulfonamides. Since the Mab had good sensitivity toward two-ring sulfonamides containing a six-membered ring at the N1

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RESULTS AND DISCUSSIONS Synthesis of Haptens. It is well-known that generation of a qualified Mab depends on delicate hapten design and extensive hybridoma screening. Moreover, the chemical structure of hapten is a primary factor for the CR of the Mab. Different hapten structures lead to different CR of the Mab. There have been many haptens reported for sulfonamide immunization in previous studies. In order to obtain a broadspecific Mab, the common sulfonamide core structure (4aminobenzensulfonylamino) cannot be changed. Wang et al.18 compared the sensitivity and selectivity of several Mabs and showed that Mab 4D11 was the best. Mab 4D11 recognized 12 sulfonamides with IC50 values ranging from 1.2 to 12.4 ng/mL. However, the sensitivity of Mab 4D11 toward other sulfonamides, such as single-ring sulfonamides (1, 2, and 3) and some two-ring sulfonamides (5, 6, 7, 8, and 21), was poor. In our previous work, three haptens were employed to synthesize immunogens. The results indicated that the hapten S1 was the best. In this study, we chose four new haptens to synthesize immunogens and all seven haptens were used to synthesize coating antigens. Mice sera were screened by seven coating antigens. In our opinion, the coating antigen is as important as the immunogen. An appropriate combination of coating antigen and immunogen determines the sensitivity and selectivity of the Mab. To obtain an antibody exhibiting the desired group specificity, similarity of the steric, hydrophobic, and electronic properties of a hapten to those of the parent molecules should be maximized.39,40 In general, the reported generic haptens can 8252

DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

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Journal of Agricultural and Food Chemistry Table 1. Cross-Reactivity of Mab 3D1 and Mabs (4D11 and 4C7) in Reference compounds

3D1 (μg/L)

CR (100%)

4D1118 (μg/L)

4C718 (μg/L)

Single-Ring Sulfonamides 1 1.89 27.0 154.2 2 2.03 25.1 717.2 3 2.14 23.8 1927.5 Two-Ring Sulfonamides Containing a Five-Membered Ring 4 2.18 23.4 0.449 5 3.11 16.4 110.14 6 15.38 3.3 140.2 7 0.53 96.2 55.1 8 0.72 70.8 30.53 Two-Ring Sulfonamides Containing a Six-Membered Ring 9 1.03 49.5 3.65 10 1.94 26.9 2.23 11 0.51 100.0 3.084 12 0.37 137.8 2.53 13 0.56 91.1 0.437 14 0.35 145.7 0.57 15 0.54 94.4 0.186 16 1.17 43.6 17.13 17 0.87 58.6 0.56 18 0.74 68.9 1.04 19 0.89 57.3 −a 20 1.65 30.9 19.3 21 1.98 25.8 45.35 22 5.25 9.7 1.81 23 0.55 92.7 0.2 Three-Ring Sulfonamides 24 0.15 340.0 0.42 25 1.98 25.8 88.11 26 2.08 24.5 −a 27 0.39 130.8 3.084 a

3411 >50 000 >50 000 6.3 1.9 1.5 2359 36 109 58.8 12.4 >50 000 >50 000 0.005 25.0 86.5 >50 000 2.1 3.0 −a >50 000 37 881 36 919.2 >50 000

Figure 6. Image of trip tests for 27 sulfonamides spiked in honey samples. The spiked concentration were as following. Sulfonamides with single ring: 1 (0, 1, 2.5, 5, 10, and 25 μg/kg), 2 (0, 5, 10, 20, and 50 μg/kg), 3 (0, 2.5, 5, 10, and 25 μg/kg). Sulfonamides with two-ring containing a five-atom-ring at N1 position: 4 (0, 0.5, 1, 2.5, and 5 μg/ kg), 5 (0, 2.5, 5, 10, 25, and 50 μg/kg), 6 (0, 10, 25, 50, and 100 μg/ kg), 7 (0, 0.5, 1, 2.5, and 5 μg/kg), 8 (0, 0.5, 1, 2.5, and 5 μg/kg). Sulfonamides with two-ring containing a six-atom-ring at N1 position: 9 (0, 0.5, 1, 2.5, and 5 μg/kg), 10 (0, 0.5, 1, 2.5, and 5 μg/kg), 11 (0, 0.5, 1, and 2.5 μg/kg), 12 (0, 0.5, 1, 2.5, and 5 μg/kg), 13 (0, 0.5, 1, 2.5, and 5 μg/kg), 14 (0, 0.25, 0.5, 1, and 2.5 μg/kg), 15 (0, 0.5, 1, 2.5, and 5 μg/kg), 16 (0, 0.5, 1, 2.5, and 5 μg/kg), 17 (0, 0.5, 1, 2.5, and 5 μg/kg), 18 (0, 0.5, 1, 2.5, and 5 μg/kg), 19 (0, 0.5, 1, 2.5, and 5 μg/ kg), 20 (0, 2.5, 5, and 10 μg/kg), 21 (0, 0.5, 1, 2.5, and 5 μg/kg), 22 (0, 1, 2.5, 5, and 10 μg/kg), 23 (0, 2.5, 5, and 10 μg/kg). Sulfonamides with three-ring: 24 (0, 0.1, 0.25, 0.5, 1, and 2.5 μg/kg), 25 (0, 5, 10, 25, and 25 μg/kg), 26 (0, 2.5, 5, 10, and 25 μg/kg), 27 (0, 0.5, 1, 2.5, 5, and 10 μg/kg).

57.7 >50 000 −a 13 468

Not detected.

15, and 4) exhibited sensitive vLOD values (below 5 μg/kg) that satisfy the needs of the market. Validation of Strip Test with Positive Honey Samples and Pork Liver Samples. The results of the strip test for positive honey and pork liver samples are shown in Figure 7. For honey samples, when diluted 20 times (20×), the concentration of 4 in honey samples was 1.85 μg/kg, which led to an obvious difference between the control and test line. Because of the sensitivity of the strip test for 4, the test line can hardly be observed when diluted 5× and 10×. For pork liver samples, because of high sensitivity of the strip test for 11, the extract can be diluted many times to eliminate matrix interference. When diluted 200×, the strip test can still be applied for the analysis. To a degree, the high sensitivity of the immunochromatographic strip test allows fast screening of complicated food samples, especially for tissue samples. In conclusion, considerable effort has been made to produce antibodies (polyclonal antibody or Mab) that can recognize one or several sulfonamides, with the aim of developing an applicable immunoassay for the fast screening of sulfonamides. On the basis of previous reports, we have used several haptens with slightly modified structures to obtain a more groupspecific and sensitive Mab. To broaden the selectivity of the Mab, the hapten with a six-membered pyrimidine ring at the

Figure 5. Comparison of Mab 3D1 and Mab 4D1.18

position, the strip tests for these sulfonamides exhibited good vLOD values. In addition, the strip tests for the remaining four sulfonamides containing three rings also exhibited good sensitivity when testing honey samples. In conclusion, the strip tests for the five most important sulfonamides (11, 24, 13, 8253

DOI: 10.1021/acs.jafc.7b03190 J. Agric. Food Chem. 2017, 65, 8248−8255

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Journal of Agricultural and Food Chemistry

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Figure 7. Image of strip tests for positive honey samples and pork liver samples. Honey samples (4): 1 = 0 (0.01 M PBS), 2 = 1.85 μg/kg (20×), 3 = 3.7 μg/kg (10×), 4 = 7.4 μg/kg (5×) . Pork liver samples (11): 1 = 0 (0.01 M PBS), 2 = 5 0.5 μg/kg (20×), 3 = 2.2 μg/kg (50×), 4 = 1.1 μg/kg (100×), 5 = 0.55 μg/kg (200×).

N1 position was preferable to a five-membered thiazole ring (hapten S1), six-membered benzene ring (hapten S4), or straight carbon chain (hapten S3). On the other hand, the heterogeneous coating antigen with a straight carbon chain (hapten S3) was better than the homogeneous coating antigen. The strip test based on the Mab was suitable for rapid detection of 27 sulfonamides by the naked eye within 5−10 min, enabling high-throughput on-site determination of sulfonamides in honey samples.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chuanlai Xu: 0000-0002-5639-7102 Funding

This work was financially supported National Key R & D Program (2016YFF0202300 and 2016YFD0401101), and grants from Natural Science Foundation of Jiangsu Province (CMB21S1614). Notes

The authors declare no competing financial interest.

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