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An ultrasensitive immunochromatographic strip for fast screening of twenty-seven 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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03190 • Publication Date (Web): 27 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017

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Page 1 of 34

Journal of Agricultural and Food Chemistry

1

An Ultrasensitive Immunochromatographic Strip for Fast

2

Screening of Twenty-seven Sulfonamides in Honey and Pork

3

Liver Samples Based on a Monoclonal Antibody

4

Yanni Chen, Lingling Guo, Liqiang Liu, Shanshan Song, Hua Kuang*, Chuanlai Xu*

5

State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, JiangSu, 214122, PRC

6

Abstract

7

Group-specific monoclonal antibodies (Mabs) with selectivity for twenty-seven

8

sulfonamides were developed based on new combinations of immunogen and coating

9

antigen. The Mab was able to recognize twenty-seven sulfonamides with 50%

10

inhibition concentration (IC50) values ranging from 0.15-15.38 µg/L. In particular, the

11

IC50

12

sulfamonomethoxine, sulfadimethoxine and sulfamethoxazole) were 0.51, 0.15, 0.56,

13

0.54, and 2.14 µg/L, respectively. Based on the Mab, an immunochromatographic

14

lateral flow strip test was established for rapid screening of sulfonamides in honey

15

samples. The visual limit of detection of the strip test for most sulfonamides in spiked

16

honey samples was below 10 µg/kg, satisfying the requirements of authorities.

17

Positive honey and pork liver samples, which had been confirmed by

18

high-performance liquid chromatography/mass spectrometry, were used to validate

19

the reliability of the proposed strip test. The immunochromatographic lateral flow

20

strip test provides a rapid and convenient method for fast screening of sulfonamides in

21

honey samples.

values

for

five

sulfonamides

(sulfamethazine,

1

ACS Paragon Plus Environment

sulfaquinoxaline,


22

Keywords: monoclonal antibody, sulfonamides, immunochromatographic lateral flow

23

strip test, honey samples, pork liver samples.

24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 2


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44

Introduction

45

Sulfonamides are widely used in veterinary medicine for treatment of infectious

46

diseases and as growth-promoting feed additives.1-3 The abuse of sulfonamides in

47

veterinary practice may lead to the presence of sulfonamide residues in foodstuffs

48

derived from animals.4 Excess sulfonamide residues are harmful to consumers

49

because of their carcinogenic potential and the risk of antibiotic resistance.5-7 In order

50

to protect consumers from these risks, the European Union and China have

51

established maximum residue limits (MRLs) for total sulfonamides in edible animal

52

tissues and milk of 100 µg/kg.8, 9 In China, the determination of five sulfonamides

53

(sulfamethazine, sulfaquinoxaline,

54

sulfamethoxazole) in meat, eggs, and milk is compulsory.8 There are currently more

55

than twenty different sulfonamides used in human and animal healthcare. Figure 1

56

shows twenty-seven sulfonamides classified according to the chemical group at the

57

N1 position of the common sulfonamide core structure.

sulfamonomethoxine,

sulfadimethoxine and

58

Chromatography and mass spectrometry are the most commonly used analytical

59

methods for detection of sulfonamides.10-13 Although these methods are sensitive and

60

specific, they require time-consuming pretreatment of samples, highly trained

61

personnel and expensive instruments. Recently, analytical methods that rely on

62

biosensors have become popular because of their high sensitivity.14-16 In general,

63

enzyme-linked immunosorbent assay (ELISA) and

64

flow strip test, both based on monoclonal antibody (Mab) are widely used for fast

65

screening in food safety assessment. Compared with the ELISA, the strip test avoids

immunochromatographic lateral

3



66

incubation and washing steps and only requires 5-10 min to fulfill the procedure.

67

Therefore, as a semi-quantitative assay, the strip test is undoubtedly more convenient

68

for on-site determination and high-throughput processing of samples. The

69

immunochromatographic strip test for small molecules is based on a competitive

70

format of indirect and competitive ELISA (Ic-ELISA). In brief, a sensitive and

71

broad-specific Mab is essential for establishment of the strip test.

72

There are numerous reports on the generation of group-specific antibodies for

73

sulfonamides, for both polyclonal antibodies and Mabs. Mabs are favored over

74

polyclonal antibodies because of their high reproducibility and unlimited supply.

75

However, the reported Mabs are generally too specific or recognize only a small

76

group of sulfonamides. For efficient surveillance, an immunoassay that can detect

77

multiple sulfonamides rather than a specific sulfonamide is preferable. In our previous

78

study, a strip test was developed based on a Mab that was able to recognize twenty-six

79

sulfonamides with IC50 values of 0.08-90.18 µg/L.17 As a consequence of the hapten

80

structure, the Mab showed better inhibition of sulfonamides containing a thiazole ring

81

at the N1 position. The sensitivities of the Mab towards sulfamethazine (18.79 µg/L)

82

and sulfaquinoxaline (39.12 µg/L) were therefore relatively poor. Recent advances in

83

the production of broad-specific Mabs for sulfonamides have been summarized by

84

Wang et al.18 The discussed Mabs recognized eight sulfonamides with IC50 values

85

below 100 µg/L. Moreover, the Mabs (4D11 and 4C7) developed by Wang’s group

86

were optimized in their further work.19 Under optimal conditions, the Mab 4D11

87

showed IC50 values for twenty-two sulfonamides at concentrations below 100 µg/L, 4


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88

demonstrating significant progress in the production of broad-specific Mabs for

89

sulfonamides. In addition, Yuan et al.20 developed a Mab that recognized sixteen

90

sulfonamides with IC50 values of 0.52-51 µg/L. Clearly, there is still scope to improve

91

the sensitivity towards some sulfonamides as well as produce broader specificity

92

Mabs. Therefore, the aim of this study was to develop Mabs with wider applicability,

93

primarily

94

immunochromatographic lateral flow strip test was established based on the proposed

95

Mabs for detection of sulfonamides in honey and pork liver samples.

96

Materials and methods

97

Reagents and apparatus

98

Sulfaceamide (1), sulfaguanidine (2), sulfanilamide (3), sulfamethoxazole (4),

99

sulfamethizole (5), sulfathiazole (6), sulfamoxol (7), sulfisoxazole (8), sulfadiazine (9)

100

sulfamerazine (10), Sulfamethazine (11), sulfasomidine (12), sulfamonomethoxine

101

(13),

102

sulfamethoxypyridazine (17), sulfachloropyridazine (18), sulfaclozine (19), sulfalene

103

(20), sulfabenzamide (21), sulfapyridine (22), sulfanitran (23), sulfaquinoxaline (24),

104

sulfaphenazole (25), sulfasalazine (26) and phthalylsulfathiazole (27) were purchased

105

from Dr. Ehrenstorfer (Augsburg, Germany) or Sigma-Aldrich (Shanghai, China).

106

Bovine

107

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and N-hydroxysuccinimide

108

(NHS), and enzyme immunoassay-grade horseradish peroxidase labelled goat

109

anti-mouse immunoglobulin (IgG) were obtained from Sigma-Aldrich.

in

respect

sulfameter

serum

to

(14),

sensitivity

and

sulfadimethoxine

albumin

specificity.

(15),

(BSA),

5


Furthermore,

sulfadoxine

ovalbumin

an

(16),

(OVA),


Page 6 of 34

110

The materials (Polyvinylchloride pads, absorbance pad, sample pad (glass-fiber

111

membrane) and nitrocellulose (NC) membrane) for strip test were obtained from JieYi

112

Biotechnology Co., Ltd. (Shanghai, China). The CM4000 Guillotine Cutting Module

113

and the Dispensing Platform, which were used to obtain individual strips and spray

114

reaction reagents, were purchased from Kinbio Tech Co., Ltd. (Shanghai, China).

115

Absorbance measurements were performed with a spectrophotometric microtiter plate

116

reader (Thermo, Waltham, MA, US), and UV spectra were determined with an

117

ultraviolet-visible spectrophotometer (Agilent, Santa Clara, CA, US).

118

Synthesis of haptens and immunogens

119

The haptens were synthesized as previously reported.18,

120

N-acetylsulfaniyl chloride was replaced by amino groups of different chemicals

121

containing carboxyl groups to obtain the following haptens (Figure 2):

122

2-(2-(4-aminophenylsulfonamido)

123

4-(4-aminophenylsulfonamido) benzoic acid (S2), 6-(4-aminophenylsulfonamido)

124

hexanoic

125

2-(4-aminophenylsulfonamido)

126

2-(4-aminophenylsulfonamido)-4-methylpyrimidine-5-carboxylic

127

(E)-5-(2-(4-aminophenylsulfonamido)-4,6-dimethylpyrimidin-5-yl) pent-4-enoic acid

128

(S7 ). The structures of seven haptens were confirmed by 1H NMR spectra.

129

Hapten S1

130

1

131

2H, J = 8.4Hz, CHar), 6.476 (s, 1H, SO2-NH), 5.840 (s, 2H, NH2), 3.469 (s, 2H, CH-

132

2-COOH).

acid

(S3),

thiazol-4-yl)

21

The chloride atom of

acetic

4-(4-(4-aminophenylsulfonamido)

acid

butanoic

pyrimidine-5-carboxylic

acid

acid acid

(S1),

(S4), (S5), (S6),

H NMR (400 MHz, DMSO-d6), δ (ppm) 7.434 (d, 2H, J = 8.8Hz, CHar ), 6.559 (d,

6


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133

Hapten S2

134

1

135

SO2-NH), 7.792 (d, 2H, J = 8.4Hz, CHar), 7.468 (d, 2H, J = 8.8Hz, CHar), 7.165 (d,

136

2H, J = 8.8Hz, CHar), 6.560 (d, 2H, J = 8.4Hz, CHar), 6.050 (s, 2H, NH2)

137

Hapten S3

138

1

139

8.8Hz, CHar), 7.047 (t, 1H, J = 12.4Hz, SO2-NH), 6.607 (dd, 2H, J = 8.8Hz, CHar ),

140

5.888 (s, 2H, NH2), 2.639-2.589 (m, 2H, NH-CH2), 2.163-2.126 (t, 2H, CH2-COOH),

141

1.431-1.184 (br, m, 6H, CH2- CH2- CH2).

142

Hapten S4

143

1

144

SO2-NH), 7.367 (d, 2H, J = 8.8Hz, CHar ), 7.023-6.951 (m, 4H, CHar), 6.526 (d, 2H,

145

J = 8.8Hz, CHar ), 5.939 (s, 2H, NH2 ), 2.476-2.438 (t, 2H, CH2 ph), 2.175-2.139 (t,

146

2H, CH2COOH), 1.724 -1.686 (t, 2H, CH2 ).

147

Hapten S5

148

1

149

8.4Hz, CHar), 6.075-6.032 (br, 2H, NH2).

150

Hapten S6

151

1

152

8.8Hz, CHar), 6.570 (d, 2H, J = 8.4Hz, CHar), 2.567-2.505 (d, 2H, CHar).

153

Hapten S7

154

1

H NMR (400 MHz, DMSO-d6), δ (ppm) 12.686 (s, 1H, COOH), 10.420 (s, 1H,

H NMR (400 MHz, DMSO-d6), δ (ppm) 11.944 (s, 1H, COOH), 7.402 (d, 2H, J =

H NMR (400 MHz, DMSO-d6), δ (ppm) 12.023 (s, 1H, COOH), 9.714 (s,1H,

H NMR (400 MHz, DMSO-d6), δ (ppm) 8.868 (s, 2H, CHar), 7.652 (d, 2H, J =

H NMR (400 MHz, DMSO-d6), δ (ppm) 8.708 (s, 1H, SO2-NH), 7.639 (d, 2H, J =

H NMR (400 MHz, DMSO-d6), δ (ppm) 7.640 (d, 2H, J = 8.8Hz, CHar), 5.945 (s, 7



Page 8 of 34

155

2H,

NH2), 5.444-5.395 (m, 2H, CH=CH ), 3.213-2.166 (m, 2H, CH2COOH), 2.876

156

(d, 2H, J = 5.6Hz, CH2), 2.328-2.274(m, 6H, CH3 ).

157

Hapten S4-S7 were respectively conjugated with BSA using EDC/NHS method to

158

obtain immunogens.22,

159

method were regarded as coating antigens. Briefly, 17.16 mg of NHS was added to a

160

solution of S4 (24.97 mg dissolved in 3 mL of methanol) with continuous stirring.

161

After 10 min, 28.51 mg of EDC was added to the solution, followed by reaction for 5

162

h at room temperature. The reaction mixture was then added to a solution of BSA (50

163

mg dissolved in 5 mL of carbonate buffer (0.05 mol/L, pH 9.6)) and stirred at room

164

temperature overnight. The solution was then dialyzed to obtain pure immunogen.

165

The other immunogens and coating antigens were prepared similarly. The

166

immunogens and coating antigens were confirmed by their UV spectra as shown in

167

Figure 3.

168

Immunization schedule

169

The procedure for immunization was similar to that used in our previous work.24, 25

170

Four immunigens (S4-EDC-BSA, S5-EDC-BSA, S6-EDC-BSA, and S7-EDC-BSA,

171

respectively) were used to immunize mice respectively. After the third immunization,

172

ic-ELISA was performed to screen the sera collected from mice. In the first analysis,

173

seven coating antigens were respectively coated on microtiter plates and seven

174

sulfonamides

175

to screen the mice serum. As a result, the appropriate coating antigen was screened.

176

After the fifth immunization, the mice with high titer and low inhibitory values for the

(5,

23

9,

All haptens conjugated with OVA using the EDC/NHS

11,

13,

14,

15

and

8


24)

were

applied

Page 9 of 34


177

seven compounds were selected for further analysis. This time twenty-seven

178

compounds were used in ic-ELISA to

179

broadest cross-reactivity was chosen as the spleen donor. Two days prior to cell fusion,

180

a final intra-peritoneal booster injection (25 µg of immunogen directly dissolved in

181

100 µL of physiological saline) was administered.

182

Cell fusion and hybridoma screening

183

The cell fusion process was performed as previously described.26,

184

screening was conducted with ic-ELISA. After three sub-clones, the best cell lines

185

were screened and cultured on a large scale. The cell lines were then intraperitoneally

186

injected into mice primed with paraffin to produce ascites. After 7-10 d, ascites were

187

collected and purified (octanoic acid-saturated ammonium sulfate method) to obtain

188

the Mabs. The concentration of Mab was determined by UV/Vis spectroscopy at 278

189

nm.

190

Ic-ELISA

191

The sensitivity and cross-reactivity of the Mabs were evaluated by ic-ELISA. Firstly,

192

the appropriate concentration of coating antigen and Mab were determined using

193

bi-dimensional titration assay. The detailed procedure was as described in the

194

literature.28, 29

identify the best mouse, and the mouse with

27

Hybridoma

195

The IC50 (concentration of competing compound that produced 50% inhibition of

196

antibody binding to the coating antigen) is considered as an important criterion for

197

evaluation of Mab sensitivity.30, 31 The IC50 values of twenty-seven sulfonamides were

198

evaluated, respectively.

199

The ability of structurally related compounds to bind to the Mab is an indicator 9



200

of specificity.28 Since the IC50 values of twenty-seven sulfonamides were determined,

201

the cross-reactivity (CR) could be calculated according to the following equation:

202

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

203

Immunochromatographic lateral flow strip test

204

Preparation of colloidal gold

205

Colloidal gold with a diameter of 20 nm was prepared

206

reduction method.23, 32, 33 Briefly, freshly prepared 1% (w/v) trisodium citrate was

207

added to boiling aqueous HAuCl4·4H2O in a flask with vigorous stirring. The mixture

208

was persistently boiled until the color of the solution changed to wine-red. The

209

solution was then cooled to room temperature and stored at 4°C for future use. The

210

colloidal gold was characterized by transmission electron microscopy.

211

Preparation of colloidal gold-Mab conjugates

212

The procedure for labelling Mab with colloidal gold is well-established in our

213

laboratory.34 In theory, the negatively charged colloidal gold can combine with the

214

positively charged groups of the Mab via electrostatic interaction, which is more

215

stable under weakly alkaline conditions. Firstly, K2CO3 (0.1 M) was used to adjust the

216

colloidal gold to pH 8. The Mab was then slowly added to the colloidal gold solution.

217

To block excess reactivity of colloidal gold, BSA dissolved in ultrapure water was

218

added to the mixture under stirring for 30 min. Centrifugation at 875 g for 40 min was

219

fulfilled to remove free blocking agent and excess Mab. The sediment was

220

re-suspended twice in borate buffer (0.002 M, pH 8, containing 1% (w/v) sucrose and

221

0.01% Tween-20).

10


using the sodium citrate

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222

The principle of strip test

223

A competitive format similar to ic-ELISA was the basis of the strip test.35, 36 The

224

assembly and principle of the strip is shown in Figure 4. After insertion into the

225

sample extract, the end of the sample pad rapidly wetted. Colloidal gold-Mab

226

immobilized on the conjugate pad is dissolved and begins to flow with the sample up

227

the NC membrane under the capillary effect. The strip is then placed flat to allow the

228

solution to transfer smoothly. Goat anti-mouse IgG immobilized on the control line

229

can capture the colloidal gold-Mab, forming a red band that certifies the validity of

230

the strip test. For negative samples, the colloidal gold-Mab can conjugate with both

231

coating antigen on the test line and goat anti-mouse IgG on the control line, meaning

232

that two red bands appear. In the contrast, for the positive samples, the limited binding

233

site on the colloidal gold-Mab are partially occupied by target analytes in the samples.

234

Therefore, the amount of coating antigen that can combine with colloidal gold-Mab is

235

reduced, resulting in a colorless band on test line than on control line. The higher the

236

concentration of analyte in sample, the less colored the test line is. If the concentration

237

of analyte in the samples is below the LOD, the colors of the two lines cannot be

238

distinguished. The concentration that leads to an obvious difference between test and

239

control lines is defined as the visual limit of detection (vLOD).

240

Analysis of spiked honey samples by strip test

241

The negative honey samples confirmed by HPLC/MS were supported by Jiangsu

242

Entry-Exit Inspection and Quarantine Bureau.37,

243

spiked with different concentration of twenty-seven sulfonamides, respectively.

244

Honey samples were diluted by phosphate buffered solution (PBS (0.01M, pH 7.2))

245

two times to eliminate matrix interference. The final concentrations of sulfonamides

38

Negative honey samples were

11



246

in honey samples was as follows: 1 (0, 1, 2.5, 5, 10, and 25 µg/kg); 2 (0, 5, 10, 20,

247

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,

248

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

249

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,

250

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,

251

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

252

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,

253

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,

254

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),

255

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,

256

10 and 25 µg/kg), 27 (0, 0.5, 1, 2.5, 5, and 10 µg/kg).

257

Analysis of positive samples

258

The positive honey samples and pork liver sample contained known concentration of

259

sulfonamides were also provided by Jiangsu Entry-Exit Inspection and Quarantine

260

Bureau.

261

The honey sample (NO.9788) contains 4 at a level of 37 µg/kg and the pork liver

262

sample contains 11 at a level of 110 µg/kg. 37, 38 Based on the spiked-recovery test for

263

4 and 11, the honey sample was diluted with 5, 10, 20 times respectively. Therefore,

264

the final concentration of 4 was 7.4, 3.7, and 1.85 µg/kg. Then, the honey samples

265

with different dilution times were analyzed by strip test.

266

20 mL of ethyl acetate was added to 5 g of pork liver sample in a 50 mL

267

centrifuge tube and subjected to vigorous shaking for 5 min. The mixture was 12


Page 12 of 34

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268

centrifuged at 875 g for 5 min and then the supernatant was collected in a new tube.

269

The sediment was extracted with 10 mL of ethyl acetate, following by centrifugation.

270

The combined supernatants were dried in a stream of nitrogen gas. 5 mL of 0.01 M

271

PBS was added to dissolve the dried extract. The dissolved extract then be diluted 20,

272

50, and 100 times with 0.01 M PBS. Therefore, the final concentration of 11 was 5.5,

273

2.2, and 1.1 µg/kg. Each of the diluted extract samples were analyzed by strip test.

274 275

Results and discussions

276

Synthesis of haptens

277

It is well known that generation of a qualified Mab depends on delicate hapten design

278

and extensive hybridoma screening. Moreover, the chemical structure of hapten is a

279

primary factor for the CR of the Mab. Different hapten structures lead to different CR

280

of the Mab. There have been many haptens reported for sulfonamide immunization in

281

previous studies. In order to obtain a broad-specific Mab, the common sulfonamide

282

core structure (4-aminobenzensulfonylamino) cannot be changed. Wang et al.18

283

compared the sensitivity and selectivity of several Mabs and showed that Mab 4D11

284

was the best. Mab 4D11 recognized 12 sulfonamides with IC50 values ranging from

285

1.2-12.4 ng/mL. However, the sensitivity of Mab 4D11 towards other sulfonamides,

286

such as single-ring sulfonamides (1, 2, and 3) and some two-ring sulfonamides (5, 6, 7

287

8 and 21), was poor. In our previous work, three haptens were employed to synthesize

288

immunogens. The results indicated that the hapten S1 was the best. In this study, we

289

chose four new haptens to synthesize immunogens and all seven haptens were used to 13



290

synthesize coating antigens. Mice sera were screened by seven coating antigens. In

291

our opinion, the coating antigen is as important as the immunogen. An appropriate

292

combination of coating antigen and immunogen determines the sensitivity and

293

selectivity of the Mab.

294

To obtain an antibody exhibiting the desired group specificity, similarity of the

295

steric, hydrophobic, and electronic properties of a hapten to those of the parent

296

molecules should be maximized.39, 40

297

divided into two groups: single-ring haptens and two-ring haptens. The single-ring

298

haptens have been proven to have limited utility for Mab production.17, 18, 41 In the

299

case of two-ring haptens, most group-specific antibodies showed relatively weak

300

sensitivity against 11, the most important individual sulfonamides. In our previous

301

work17, Mab generated by hapten S1 exhibited good inhibition for sulfonamides

302

containing a thiazole ring and relatively weak inhibition for sulfonamides with

303

six-membered ring at N1 position, especially for 11. Mab generated by hapten S2,

304

containing a benzene ring at the N1 position, showed no inhibition for any

305

sulfonamides. We postulated that the two-carbon alkyl chain in the para-position of

306

the benzene ring might be too short, resulting in the benzene ring being hidden by the

307

carrier protein. Therefore, the alkyl chain of hapten S2 was extended to be a

308

four-carbon atom in hapten S4. However, mice sera still showed no inhibition against

309

any sulfonamides. The two-ring haptens containing a benzene ring were rejected

310

based on the results with haptens S2 and S4. We then chose two-ring haptens

311

containing a pyrimidine ring (haptens S5, S6, and S7). Compared with the structure of

In general, the reported generic haptens can be

14


Page 14 of 34

Page 15 of 34


312

hapten S5, hapten S6 has a extra side chain and hapten S7 has a longer alkyl chain as

313

well as two side chains. The sera of mice immunized with hapten S5 showed

314

inhibition against most of the sulfonamides using the heterogenous coating antigen

315

(hapten S3). For hapten S6, we considered that the alkyl chain was too short and that

316

the methyl group on the pyrimidine ring introduced steric hindrance, preventing

317

exposure of the antigenic determinant. The sera of mice immunized with hapten S7

318

showed good inhibition only for 11. In the case of hapten S7, regardless of the two

319

side chains, the alkyl chain was sufficiently long to expose the antigenic determinant.

320

Furthermore, the pyrimidine ring with two methyl groups was a structural fragment of

321

11. Therefore, the mice immunized with hapten S7 showed good inhibition for 11.

322

Screening of anti-sera

323

The sera resulting from different haptens were assayed by ic-ELISA with one

324

homogeneous and six heterogeneous coating antigens. The results showed that the

325

combination of hapten S5 (immunogen) and hapten S3 (coating antigen) was optimal.

326

Cross-reactivity of Mab

327

The CR of Mab 3D1 was shown in Table 1. As discussed in the section of hapten

328

synthesis, the IC50 values for sulfonamides containing a six-atom pyrimidine ring

329

were low, due to the structure of hapten S5. Moreover, for sulfonamides containing

330

pyrazine or pyridazine rings (isomers of pyrimidine ring), the IC50 values were also

331

good. Recognition of sulfonamides containing a five-membered ring by Mab 3D1 was

332

relatively weak, especially for 6. However, the vLOD of strip test for 6 was still

333

sufficient to satisfy the MRLs of European Union. The CR data of the reported Mabs 15



334

4D11 and 4C718 are also listed in Table 1. Moreover, a comparison of Mab 3D1 and

335

the previously reported Mab 4D11 is shown in Figure 5. It is apparent that the

336

sensitivity and CR of Mab 3D1 were superior to those of Mab 4D11.

337

Analysis of honey samples spiked with sulfonamides

338

Strip tests were used to analyze honey samples spiked with sulfonamides. As shown

339

in Figure 6, the vLOD of the three sulfonamides containing a single-ring were 2.5, 10,

340

and 5 µg/kg. With respect to sulfonamides containing a five-membered ring at N1

341

positon, the Mab 3D1 has a weaker sensitivity against 6. Therefore, the vLOD of the

342

strip test for 6 was 25 µg/kg, which was higher than for other sulfonamides.

343

Since the Mab had good sensitivity towards two-ring sulfonamides containing a

344

six-membered ring at the N1 position, the strip tests for these sulfonamides exhibited

345

good vLOD values. In addition, the strip tests for the remaining four sulfonamides

346

containing three rings also exhibited good sensitivity when testing honey samples. In

347

conclusion, the strip tests for the five most important sulfonamides (11, 24, 13, 15,

348

and 4) exhibited sensitive vLOD values (below 5 µg/kg) that satisfy the needs of the

349

market.

350

Validation of strip test with positive honey samples and pork liver samples

351

The results of the strip test for positive honey and pork liver samples are shown in

352

Figure 7. For honey samples, when diluted 20 times (20X), the concentration of 4 in

353

honey samples was 1.85 µg/kg, which led to an obvious difference between the

354

control and test line. Due to the sensitivity of the strip test for 4, the test line can

355

hardly be observed when diluted 5X and 10X. For pork liver samples, because of high 16


Page 16 of 34

Page 17 of 34


356

sensitivity of strip test for 11, the extract can be diluted many times to eliminate

357

matrix interference. When diluted 200X, the strip test can still be applied for the

358

analysis. To a degree, the high sensitivity of immunochromatographic strip test allows

359

fast screening of complicated food samples, especially for tissue samples.

360

Conclusion

361

Considerable effort has been made to produce antibodies (polyclonal antibody or Mab)

362

that can recognize one or several sulfonamides, with the aim of developing an

363

applicable immunoassay for the fast screening of sulfonamides. Based on previous

364

reports, we have used several haptens with slightly modified structures to obtain a

365

more group-specific and sensitive Mab. To broaden the selectivity of the Mab, the

366

hapten with a six-membered pyrimidine ring at the N1 position was preferable to a

367

five-membered thiazole ring (hapten S1), six-membered benzene ring (hapten S4) or

368

straight carbon chain (hapten S3). On the other hand, the heterogenous coating

369

antigen with a straight carbon chain (hapten S3) was better than the homogenous

370

coating antigen. The strip test based on the Mab was suitable for rapid detection of

371

twenty-seven sulfonamides by the naked eye within 5-10 min, enabling

372

high-throughput on-site determination of sulfonamides in honey samples.

373 374

Compliance with Ethical Standards

375

Author Information

376

*Corresponding author: E-mail: [email protected]; [email protected]

377 378

Acknowledgements 17



379

This

work

was

financially

supported

National Key R

Page 18 of 34

&

D Program

380

(2016YFD0501208, 2016YFF0202300).

381

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Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting

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19-nortestosterone residue in animal tissues. Journal of agricultural and food

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Liquid Chromatography-High-Resolution Mass Spectrometry. Journal of agricultural

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and food chemistry 2017, 65, 1984-1991.

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39. Mercader, J. V.; Agullo, C.; Abad-Somovilla, A.; Abad-Fuentes, A., Synthesis of

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site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

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Org. Biomol. Chem. 2011, 9, 1443-1453.

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40. Spinks, C. A.; Wyatt, G. M.; Lee, H. A.; Morgan, M. R. A., Molecular modeling

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of hapten structure and relevance to broad specificity immunoassay of sulfonamide

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antibiotics. Bioconjugate Chem. 1999, 10, 583-588.

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41. Zhang, H. Y.; Duan, Z. J.; Wang, L.; Zhang, Y.; Wang, S., Hapten synthesis and

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21


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development of polyclonal antibody-based multi-sulfonamide immunoassays. Journal

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of agricultural and food chemistry 2006, 54, 4499-4505.

520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539

Figure Captions

540

Figure 1. Chemical structures of twenty-seven sulfonamides. The sulfonamides are 22


Page 22 of 34

Page 23 of 34


541

arranged by the number of rings and the number of atoms in the ring at the N1

542

position.

543

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

544

Figure 3. The UV/Vis spectra of immunogen and coating antigen.

545

Figure 4. Schematic illustration of the immunochromatographic lateral flow strip test.

546

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

547

Figure 6. Image of trip tests for twenty-seven sulfonamides spiked in honey samples.

548

The spiked concentration were as following: sulfonamides with single ring : 1 (0, 1,

549

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

550

µg/kg); sulfonamides with two-ring containing a five-atom-ring at N1 position: 4 (0,

551

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

552

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

553

with two-ring containing a six-atom-ring at N1 position: 9 (0, 0.5, 1, 2.5, and 5 µg/kg),

554

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

555

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,

556

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),

557

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

558

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,

559

and 10 µg/kg), sulfonamides with three-ring: 24 (0, 0.1, 0.25, 0.5, 1, and 2.5 µg/kg),

560

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,

561

and 10 µg/kg).

562

Figure 7. Image of strip tests for positive honey samples and pork liver samples. 23



563

Honey samples (4): 1=0 (0.01 M PBS), 2=1.85 µg/kg (20X), 3=3.7 µg/kg (10X),

564

4=7.4 µg/kg (5X) . Pork liver samples (11): 1=0 (0.01 M PBS), 2=5 .5 µg/kg (20X),

565

3=2.2 µg/kg (50X), 4=1.1 µg/kg (100X), 5=0.55 µg/kg (200X).

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583

Tables:

584

Table 1. Cross-reactivity of Mab 3D1 and Mabs (4D11 and 4C7) in Reference

585

a

Not detected 24


Page 24 of 34

Page 25 of 34


Compounds

3D1

CR

4D1118

4C718

(µg/L)

(100%)

(µg/L)

(µg/L)

Single-ring sulfonamides 1

1.89

27.0

154.2

3411

2

2.03

25.1

717.2

> 50,000

3

2.14

23.8

1927.5

> 50,000

Two-ring sulfonamides containing a five-membered ring 4

2.18

23.4

0.449

6.3

5

3.11

16.4

110.14

1.9

6

15.38

3.3

140.2

1.5

7

0.53

96.2

55.1

2359

8

0.72

70.8

30.53

36109

Two-ring sulfonamides containing a six-membered ring 9

1.03

49.5

3.65

58.8

10

1.94

26.9

2.23

12.4

11

0.51

100.0

3.084

> 50.000

12

0.37

137.8

2.53

> 50.000

13

0.56

91.1

0.437

0.005

14

0.35

145.7

0.57

25.0

15

0.54

94.4

0.186

86.5

16

1.17

43.6

17.13

> 50.000

17

0.87

58.6

0.56

2.1

18

0.74

68.9

1.04

3.0

a

-a

19

0.89

57.3

-

20

1.65

30.9

19.3

> 50.000

21

1.98

25.8

45.35

37,881

22

5.25

9.7

1.81

36919.2

23

0.55

92.7

0.2

> 50.000

Three-rings sulfonamides 24

0.15

340.0

0.42

57.7

25

1.98

25.8

88.11

> 50.000

a

26

2.08

24.5

27

0.39

130.8

-

3.084

586

25


-a 13,468


587 588 589 590 591 592 593 594 595 596 597 598 599 600

26


Page 26 of 34

Page 27 of 34


601 602

Figure 1

603 604 605 606 607 608 609 610 611 612

27



613 614

Figure 2

615 616 617 618 619 620 621 622 623 624 625 626 627

28


Page 28 of 34

Page 29 of 34


628 629

Figure 3

630 631 632 633 634 635 636 637 638 639 640

29



641 642 643 644 645 646 647 648 649 650 651 652 653 654

Figure 4

30


Page 30 of 34

Page 31 of 34


655 656

Figure 5

657 658

31



659 660

Figure 6.

661 662 663 664 665 666 667 668 32


Page 32 of 34

Page 33 of 34


669 670 671

672 673

Figure 7

674 675 676 677 678 679 680 681 682 683 684 685 33



686

687 688

Table of Contents Graphic

34


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Ultrasensitive Immunochromatographic Strip for Fast Screening of 27

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