Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional

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Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional Multianalyte Immunoassay for 31 #-agonists and Analogues Lanteng Wang, Wen Meng Jiang, Xing Shen, Xiangmei Li, Xinan Huang, Zhenlin Xu, Yuanming Sun, Shun-Wan Chan, Lingwen Zeng, Sergei Alexandrovich Eremin, and Hongtao Lei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04684 • Publication Date (Web): 21 Jan 2018 Downloaded from http://pubs.acs.org on January 21, 2018

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Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

1

Four Hapten Spacer Sites Modulating Class Specificity:

2

Non-directional Multianalyte Immunoassay for 31 β-agonists and

3

Analogues

4

Lanteng Wanga #, Wenmeng Jianga #, Xing Shena, Xiangmei Lia, Xin-an Huangb,

5

Zhenlin Xua, Yuanming Suna, Shun-Wan Chanc, Lingwen Zengd, Sergei

6

Alexandrovich Eremine, f, Hongtao Leia*

7 8

a

9

Agricultural University, Guangzhou 510642, China

Guangdong Provincial Key Laboratory of Food Quality and Safety, South China

10

b

11

Innovation Center, Guangzhou University of Chinese Medicine,

12

Guangzhou 510405, China

13

c

14

Hong Kong, Hong Kong, China

15

d

16

Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou

17

510530, China

18

e

19

Russia

20

f

21

Russian Academy of Sciences, 119071 Moscow, Russia

22

* Corresponding author. Phone: +8620 8528 3925. Fax: +8620 8528 0270. E-mail:

Tropical Medicine Institute & South China Chinese Medicine Collaborative

Faculty of Science & Technology, Technology & Higher Education Institute of

South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou

Faculty of Chemistry, M.V. Lomonosov Moscow State University, 119991 Moscow,

A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23 24

[email protected]. #

Wang L. and Jiang W. contributed equally to this work.

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Abstract: Immunoassay methods are important to monitor β-agonists illegally used

26

for reducing animal fat deposition in livestock. However, there is no a simultaneous

27

screening surveillance immunoassay for possibly occurred various β-agonist

28

chemicals in food. In this study, using an R-(–)-salbutamol derivative as the

29

immunizing hapten, an antibody recognizing 31 β-agonists and analogues was

30

generated for the first time. Three-dimensional quantitative structure-activity

31

relationship (3D QSAR) revealed that strong steric and hydrophobic fields around the

32

hapten spacer near C-2, as well as a chirality at C-1´, dominantly modulated the class

33

specificity of the raised antibody. However, a hapten spacer linked at C-2´ or C-1

34

would lead to a narrow specificity, and the spacer charge at C-6 could affect the raised

35

antibody specificity spectrum. A class specificity competitive indirect enzyme linked

36

immunosorbent assay (ciELISA) was established with an ideal recovery ranged from

37

81.8% to 118.3% based on the obtained antibody. With a good agreement to

38

HPLC-MS method, the proposed ciELISA was confirmed to be reliable for the rapid

39

surveillance screening assay of β-agonists in urine sample. This investigation will

40

contribute to the rational design and control of the immunoassay specificity.

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β-agonists are a class of therapeutic drugs commonly used in the treatments for

42

acute symptoms of asthma owing to their bronchodilator activities, e.g. salbutamol,

43

clenbuterol, terbutaline and salmeterol etc.1 An overdose of β-agonists could lead to

44

symptom of nausea, dizziness and palpitation, even cause to death.2 However, some

45

of these compounds were sometimes found to be illegally used as broncho-dilating

46

agents, because they can reduce carcass fat content and then result in a muscle

47

hypertrophy.3

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well-known as “lean meat agents” in China, these agents ever resulted in serious

49

public health accidents.4 In 2009 and 2011, a number of β-agonists poisoning

50

incidents occurred in China, and these incidents caused sickness to 70 people in

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Guangzhou and 91 people in Changsha, respectively.5 In European Union (EU) and

52

China, the illegal use of β-agonists as a growth promotor in animal husbandry is

53

forbidden.6

Salbutamol,

clenbuterol,

brombuterol

and

ractopamine

were

54

Antibody-based immunoassays are effective tools for either quantitative or

55

qualitative detection of chemical residues in foods and environment, which do not

56

require complicated equipment, and are capable of analyzing a quantity of samples

57

simultaneously.

58

immunosorbent

59

immunochromatographic test and so on, had focused on the detection of single

60

β-agonist7-8 or a limited numbers of β-agonists.9-11 However, a numerous of β-agonist

61

have been be found to ever or possibly abused in animal products.12 It would be

62

urgent to develop an immunoassay for the simultaneous detection of a series of

Most

of

immunoassay

methods,

assay

(ELISA),

DNA

4


such

as

Enzyme-linked

labeled-immunoprobe,

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63

β-agonists and analogues instead of one assay for each specific target. However, there

64

is not yet a multianalyte immunoassay for a non-directional screening purpose to

65

variously enough β-agonists and possibly occurred analogues in animal food. This is

66

at least ascribed to the lack of a class specificity antibody and understanding the class

67

recognition structure-activity relationship, and thus leading to the difficult artificial

68

control of the resultant antibody specificity.13

69

Salbutamol possesses two enantiomers, R-(–)-salbutamol and S-(+)-salbutamol.

70

R-(–)-isomer is approximately 80 times more potent than S-(+)-isomer on the

71

therapeutically bronchodilating effects, and pure R-(–)-salbutamol can reduced side

72

effects.14-15 In our previous study, the raised antibody against Rac-salbutamol

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demonstrated an unexpected cross-reactivity (CR, 447.3% and 255.8%) to

74

brombuterol and clenbuterol.16 This suggested that salbutamol would possibly be a

75

hapten candidate to produce class specificity antibody to β-agonists.

76

In this study, to develop a class specific immunoassay and better investigate the

77

recognition between the antibody and β-agonists, the pure isomer R-(–)-salbutamol

78

instead of Rac-salbutamol was selected as immunizing hapten to raise the antibody. It

79

was interestingly found that 31 β-agonists and analogues could be well recognized by

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this resultant antibody. Based on the cross-reactivity and the structure analysis, a

81

comparative molecular field analysis (CoMFA) and a comparative molecular

82

similarity indices analysis (CoMSIA) were then used for the investigation of

83

three-dimensional quantitative structure-activity relationship (3D QSAR) of the

84

antibody and β-agonists. The effect of hapten spacer sites on the class specificity were

5



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reasonably elaborated and predicted.

86 87

EXPERIMENTAL SECTION

88

Reagents and Animals. Rac-salbutamol, R-(–)-salbutamol and S-(+)-salbutamol

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were obtained from Yunhui Trade Co., Ltd., (Guangzhou, China). 3-dehydroxy

90

salbutamol (3D-SAL), pirbuterol, zilpaterol, octopamine, colterol, bitolterol,

91

indacaterol, olodaterol, fenspiride, reproterol, t-butylnorsynephrine, bromoclenbuterol,

92

benzaldehyde,5-[2-[(1,1-dimethylethyl)amino]-1-hydroxyethyl]-2-hydroxy

93

salbutamol methyl ether, salbutamon, hydroxymethyl clenbuterol (HYD), clenhexerol,

94

5-hydroxy salbutamol, 1-(3,5-dimethoxyphenyl)-2-(isopropylamino)ethanol (DIM),

95

cimbuterol and procaterol were purchased from TRC (Toronto, Canada). Salmeterol,

96

vilanterol, mabuterol, mapenterol, carbuterol, ritodrine, orciprenaline, clenbuterol,

97

clenpenterol, clenproperol and bambuterol were purchased from Dr. Ehrenstorfer

98

(Augsburg, Germany). Penbutolol, brombuterol, terbutaline, tulobuterol, ractopamine,

99

clorprenaline,

hexoprenaline

(DAH),

and

100

benzenemethanol,4-amino-α-[[(1,1-dimethylethyl)amino]methyl]

101

purchase from Witega (Berlin, Germany). Alprenolol, pindolol, cimaterol,

102

arformoterol, propafenone and propranolol were purchased from Aladdin (Shanghai,

103

China). Epinephrine, norepinephrine and isoproterenol were purchased from Kewei

104

Co., Ltd., (Shanghai, China). R-(–)-2-tert-butylamino-1-phenylethanol (R-(–)-TER),

105

S-(+)-2-tert-butylamino-1-phenylethanol

106

(DMF), isobutyl chloroformate, bovine serum albumin (BSA), ovalbumin (OVA) and

(S-(+)-TER),

6


(BEN)

were

N,N-dimethylformamide

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107

complete and incomplete Freund’s adjuvants were purchased from Sigma (St. Louis,

108

MO, USA). Phosphate-buffered saline with 0.1% Tween-20 (PBST, 0.01 mol L-1, pH

109

7.4) was for the use of the working buffer for aqueous standard solutions of analytes.

110

All other chemicals and organic solvents that were analytical grade or better were

111

obtained from a local chemical supplier (Yunhui Trade Co., Ltd., Guangzhou, China).

112

New Zealand white rabbits with 2-3 months old (about 2 kg) were purchased from

113

Guangdong Experimental Animal Center.

114

Apparatus. Ultraviolet−visible (UV−vis) spectrum was recorded on UV-4000

115

spectrophotometer (Hitachi, Japan). Electronic circular dichroism (ECD) spectrum

116

was obtained on Chirascan circular dichroism spectrometer (Applied Photophysics,

117

UK). The purified antibody concentration was determined by NanoDrop 2000c

118

spectrophotometer (Thermo Scientific, USA). ELISA plates were washed in a MK2

119

microplate washer (Thermo Scientific, USA). ELISA absorbance was measured at a

120

wavelength of 450 nm with Multiskan MK3 microplate reader (Thermo Scientific,

121

USA). Nuclear magnetic resonance (NMR) spectrum was obtained from DRX-400

122

NMR spectrometer (Bruker, Germany). The chromatography was manipulated on the

123

HPLC-MS system (LC-30-API5500, Shimadzu, Japan), and an Inertsil® ODS-SP

124

HPLC column (C18, 4.6×150 mm, 5 µm, GL science, Japan) was used.

125

Hapten Synthesis. Succinic anhydride (77 mg, 0.77 mmol) was added to a

126

stirred mixture of R-(–)-salbutamol (220 mg, 0.77 mmol) in dry ethanol (40 mL)

127

under nitrogen at room temperature. The solution was stirred at room temperature for

128

12 hours, and filtered to obtain the hapten R-(–)-salbutamol (R-(–)-sal-hapten, 39 mg,

7



129

11.5%). 1H-NMR (DMSO, 400 MHz): δ (ppm) 1.12 (9H, s), 2.38-2.41 (2H, m),

130

2.53-2.56 (2H, m), 2.76-2.78 (2H, m), 4.61-4.62 (1H, m), 5.05 (2H, d, J = 2.0 Hz ),

131

6.77 (1H, d, J = 8.0 Hz ), 7.10 (1H, dd, J = 8.4, 2.0 Hz), 7.27 (1H, d, J = 2.0 Hz ). MS

132

m/z (+ESI): 340.1 [M+H]+.

133

Antibody Production. The hapten-protein conjugates preparation and antibody

134

production methods were described in Supporting Information. The antibody from

135

rabbits were purified with caprylic acid-saturated ammonium sulfate precipitation,

136

and the purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel

137

electrophoresis (SDS-PAGE).17

138

ciELISA Procedure. The competitive indirect enzyme linked immunosorbent

139

assay (ciELISA) for β-agonists was conducted according to the previously reported

140

procedure.18 The ciELISA calibration curves were fitted with a four-parameter logistic

141

function.19 IC50 is the concentration of the analyte that resulted in 50% inhibition. The

142

limit of detection (LOD) was defined as the concentration of analyte inhibited 10%

143

binding (IC10).20 The linear range was defined as the lower and upper concentration

144

that provided 20% ~ 80% inhibition.21 The antibody specificity was measured with

145

the cross-reactivity (CR) of the structurally related β-agonists (Figure 1).22

146

Molecular Modeling. The molecular modeling was conducted by SYBYL-X

147

2.1.1 program package.23 Tripos force field and Gasteiger-Huckel charges were used

148

in the energy minimization. Calculated oil/water partition coefficient (ClogP) values

149

were computed by CLOGP program. The detailed methods were described in

150

Supporting Information.

8


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151

Sample analysis. In order to evaluate the accuracy and precision of the

152

immunoassay, the negative swine urine samples gifted by Dr. Yu Wang from

153

Guangzhou Institute for Food Control, China, were spiked with salbutamol,

154

clenbuterol, brombuterol and cimbuterol at three concentrations (1, 5 and 10 ng mL-1),

155

respectively, and then centrifuged at 27 000 g for 15 min, the supernatants were

156

collected as spiked samples for the ciELISA analysis.

157

The methods confirmation experiments were carried out by comparing ciELISA

158

with HPLC-MS. The HPLC-MS analysis of spiked urine samples was conducted

159

following the procedure of China National Standard (GB/T 22286-2008).

160 161

RESULTS AND DISCUSSION

162

Absolute configuration of hapten. The experimental ECD spectrum of the

163

R-(–)-hapten in acetonitrile was showed in Figure S1. The energy, oscillation and

164

rotational strength of R-(–)-isomer structure were calculated using time-dependent

165

density functional theory (TD-DFT) by Gaussian 16.24 And the calculated ECD

166

spectrum

167

B3LYP/6-311+G(2d,p) optimized geometries. It was found that the experimental

168

spectrum was similar to the calculated spectrum, and both spectra exhibited strong

169

positive Cotton effects near 203 nm and negative Cotton effects near 192 nm (Figure

170

S1). Therefore, the hapten absolute configuration could be confirmed as

171

R-(–)-isomer.25

172

was

computed

at

the

B3LYP/6-311+G(2d,p)

level

using

the

Immunoreagent preparation. UV−vis spectra of hapten-protein conjugate,

9



173

carrier protein (BSA, OVA) and the R-(–)-sal-hapten (Figure S2) implied that the

174

coupling of hapten and carrier protein was successful.26 The SDS-PAGE (Figure S3)

175

showed that the purified R-(–)-salbutamol antibody exhibited a heavy chain at about

176

50 kDa, and a light chain at about 25 kDa. This indicated that the antibody purity was

177

ideal for the further investigation.27 The purified antibody concentration determined

178

using A280 absorbance measurement was 3.8 mg mL-1.28

179

ciELISA. Optimal concentrations of coating antigen and antibody were critical

180

to the sensitivity of ciELISA.29-30 It was found that the combination of the coating

181

antigen at 12.5 ng mL-1 together with the antibody at 0.1 µg mL-1 (1/32000 dilution)

182

exhibited a maximum absorbance around 1.0 and an optimal sensitivity.31 The

183

established ciELISA calibration curve for R-(–)-salbutamol exhibited IC50 of 0.5 ng

184

mL-1, the concentration range from 0.1 to 40.9 ng mL-1 and the LOD of 0.04 ng mL-1

185

(Figure 2).

186

Specificity. In the United States, only ractopamine and zilpaterol were approved

187

as feed additives for cattle and swine by FDA, and other compounds are banned in

188

animal products.32 However, all β-agonists have been banned for animal products in

189

European Union (EU) and China. Ractopamine, clenbuterol, salbutamol, zilpaterol,

190

cimaterol, terbutaline and tulobuterol were officially banned, and most often found

191

being illegally used in China.5, 32 For purpose of investigating the impact of hapten

192

structure on the antibody specificity, 54 compounds were used for the evaluation of

193

cross-reactivity. These compounds included the most commonly β-agonists and their

194

analogues, e.g. clenbuterol, ractopamine, zilpaterol, clenhexerol and 3-dehydroxy

10


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195

salbutamol. Moreover, some potentially abused compounds in animal products, e.g.

196

brombuterol, bambuterol, penbuterol, clorprenaline, mabuterol and cimaterol, were

197

also included in the 54 compounds.33-34 The cross-reactivity to R-(–)-salbutamol was

198

set as the reference cross-reactivity (100%), it was found that there were 31

199

compounds could be recognized by the antibody against R-(–)-salbutamol (Table 1).

200

Comparing the salbutamol hapten structures reported previously,4, 30, 35-36 it could

201

be found that there were other four salbutamol derivatives with different spacer for the

202

antibody generation, herein named sal-hapten-1,30 sal-hapten-2,4 sal-hapten-3,35

203

sal-hapten-436 (Figure 1 & Table 1). Both sal-hapten-1 and sal-hapten-2 had a spacer

204

at a same site (C-6), and their spacer difference was one benzene ring on sal-hapten-2

205

spacer. However, the antibody against sal-hapten-1 exhibited a poorer recognition to

206

terbutaline and clenbuterol (CR < 0.1%), but the antibody against sal-hapten-2

207

showed a better recognition (CR, 10% and 107%), it could be inferred that the

208

supplement benzene ring at C-6 spacer on sal-hapten-2 contributed to the better

209

antibody binding affinity. But both resultant antibodies were still a narrow specific

210

(not class specificity) due to the limited recognition numbers.

211

Different from the same spacer sites of sal-hapten-1 and sal-hapten-2, the spacer

212

sites of sal-hapten-3 and sal-hapten-4 were derived at C-2´ and C-1, respectively.

213

Sal-hapten-3 possessed a two carbons spacer similar to that of sal-hapten-1, and

214

sal-hapten-4 had a longer spacer. It could be found that both resultant antibodies

215

raised from sal-hapten-3 and sal-hapten-4 recognized only salbutamol, still a similar

216

narrow specificity. This suggested that the hapten with a spacer at site C-2´ and C-1

11



217

might be not suitable for a class specificity generation.

218

In the present investigation, the R-(–)-sal-hapten spacer was derived at C-2 ,

219

different from other three hapten spacer sites C-2´, C-1 and C-6 of previous four

220

reported sal-hapten 1-4, and the obtained antibody resulted from R-(–)-sal-hapten

221

could recognize 31 compounds within the tested 54 β-agonists and analogues (Table

222

1), this was the most class specificity to β-agonist compounds reported so far. Based

223

on the comparison of hapten structures and specificities above, it could be found that

224

these four hapten spacer sites C-2´, C-1, C-6 and C-2 could significantly differed the

225

specificity of the resultant antibody to β-agonist compounds.

226

3D QSAR analysis. In order to better understand the effect of hatpen spacer

227

sites on the antibody specificity to β-agonists, two 3D QSAR methods CoMFA and

228

CoMSIA were conducted for the structure-activity analysis.

229

Generation of 3D QSAR Models. The interactions between the antibody and

230

β-agonists were analyzed by CoMFA and CoMSIA models, and simulated the binding

231

process from electrostatic, steric and hydrophobic fields, respectively (Table S1). In

232

the CoMFA model, the q2 and r2 values were 0.560 and 0.972. The q2 and r2 of the

233

CoMSIA model were 0.530 and 0.978. All the parameters could prove the simulation

234

result was reliable. The scatter plots of the predicted value versus experimental

235

activity were shown in Figure S6 and the values of training set and test set molecules

236

were shown in Table S2. Since the activities could be well predicted by the two

237

models, this confirmed the enough accuracy of the models.

238

The contour maps reflected the effect of different group or structure on the

12


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239

molecular binding affinity in various force fields. In CoMFA model, from the view of

240

steric field (Figure 3a), green contours meant that bulk steric groups around this area

241

could improve the biding affinity, and yellow contours would exerted a reverse effect.

242

From the view of electrostatic field (Figure 3c), blue contours meant that positive

243

charges were favorable to a high affinity, and red contours would exert a reverse

244

effect. In CoMSIA model, from the view of hydrophobic field, magenta contours

245

represented that hydrophobicity increasing was favorable to a high affinity, and white

246

contours had a reverse effect (Figure 3e).

247

C-2´ Effect. The tertamyl at C-2´ in clenpenterol (39) was surrounded by a large

248

block of green and magenta contour (Figure 3a, 3f), and this suggested that a bulky or

249

hydrophobic substituent could be favored for the antibody biding. HYD (37) showed

250

low cross-reactivity (CR, 0.3%), this could be ascribed its unfavorable hydrophilicity

251

of the hydroxyl at C-2´ (ClogP, 1.392).

252

The steric field of tertamyl on clenpenterol (39) was larger than that of tert-butyl

253

on clenbuterol (36) or isopropyl on clenproperol (40) (Figure 1), and the

254

hydrophobicity (ClogP, 2.922) of tertamyl on clenpenterol was also higher than that of

255

the tert-butyl on clenbuterol (ClogP, 2.393) and the isopropyl on clenproperol (ClogP,

256

1.994). Thus, the cross-reactivity difference (12.8% for clenperterol, 2.9% for

257

clenbuterol and ND for clenproperol) was ascribed to the difference of both the steric

258

and hydrophobic fields at C-2´ of these compounds. Similarly, it was also ascribed to

259

the steric and hydrophobic field effects that six groups of molecules demonstrate

260

cross-reactivity difference. I) t-butylnorsynephrine (16) (CR, 571.4%) and

13



261

octopamine (17) (CR, ND); II) cimbuterol (47) (CR, 4.4%) and cimaterol (48) (CR,

262

0.1%); III) mapenterol (43) (CR, 1.9%) and mabuterol (44) (CR, ND); IV) terbutaline

263

(11) (CR, 8.3%) and orciprenaline (12) (CR < 0.1%); V) colterol (7) (CR, 0.2%) and

264

isoproterenol (8) (CR, ND); VI) tulobuterol (45) (CR, 1.7%) and clorprenaline (46)

265

(CR, ND).

266

The yellow contour near C-2´ implied that a huge steric field on the tertamyl

267

would decrease the cross-reactivity. Thus, it was reasonable that those compounds

268

with bulky rings at C-2´ (e.g. arformoterol (29) (CR < 0.1%), olodaterol (31),

269

indacaterol (32), fenspiride (20), reproterol (22), ractopamine (24) and ritodrine (25)

270

(CR, ND)) demonstrated very low cross-reactivity even no recognition.

271

CoMFA analysis revealed that a stronger hydrophobic field near C-2´ could

272

increase cross-reactivity, but a huger steric field near C-2´ could decrease

273

cross-reactivity conversely. Clenhexerol (38) contained a bigger and stronger

274

hydrophobic tertiary hexyl at C-2´ (ClogP, 3.451) than the tertamyl on clenpenterol

275

(ClogP, 2.922) (Figure 1), however, its cross-reactivity was lower (CR, 9.7%) than

276

clenpenterol (CR, 12.8%). Similarly, salmeterol (35) owned a stronger hydrophobic

277

long carbon chain at C-2´ (ClogP, 3.063), its cross-reactivity (CR, 0.1%) was lower

278

than that of the weaker hydrophobic compounds R-(–)-TER (19) (ClogP, 1.816, CR,

279

4.7%) and S-(+)-TER (18) (ClogP, 1.816, CR, 0.4%). This suggested that the steric

280

and hydrophobic fields could exert effect together at C-2´, but the steric field played a

281

dominating role in the specificity modulation at C-2´.

282

CoMFA analysis also revealed that a bigger steric field at C-2´ than the isopropyl

14


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283

(e.g. the tert-butyl and tertamyl) could be conducive to a class specificity generation,

284

but the specificity would decline when the steric field continued to become larger (e.g.

285

the tertiary hexyl on clenhexerol). Compared the tert-butyl of R-(–)-sal-hapten with

286

the long spacer of sal-hapten-3, although the tert-butyl showed a smaller steric field

287

than sal-hapten-3 spacer, R-(–)-sal-hapten leaded to a better antibody binding to

288

clenbuterol (Table 1). Therefore, it suggested that the steric field of tert-butyl at C-2´

289

was beneficial to the cross-reactivity improvement. Moreover, the exposure of the

290

tert-butyl at C-2´ on the hapten was critical to obtain a class specific antibody.

291

Because one of the most important epitopes C-2´ of the hapten could not be exposed,

292

the raised antibody from sal-hapten-3 might have low cross-activity to a large

293

proportion of β-agonists and analogues with the hydrophobic groups at C-2´.

294

C-1 Effect. There were red and blue contours of electrostatic field near C-1

295

(Figure 3d), this suggested that hapten spacers connecting to site C-1 would damage

296

the electrostatic field around this site and would decrease specificity of the raised

297

antibody. Due to the electron donor inducing effect, the negative charge oxygen of the

298

phenolic hydroxyl at C-1 on R-(–)-salbutamol (3) (Gasteiger-Huckel charge, -0.322)

299

was completely exposed to the red contour, and the hydrogen with positive charge

300

(Gasteiger-Huckel charge, 0.258) was exposed to the blue contour (Figure 3d).

301

However, the negative charge nitrogen (Gasteiger-Huckel charge, -0.329) at C-1

302

amino group on clenbuterol (36) was surrounded by two positive charge hydrogens

303

(Gasteiger-Huckel charge, 0.182), this would stunt the nitrogen exposed to the red

304

contour (Figure 3c). Because of the exposed charge difference, the cross-reactivity of

15



305

clenbuterol (CR, 2.9%) became much lower than that of R-(–)-salbutamol (CR,

306

100%).

307

QSAR analysis suggested that the exposure of the hydroxyl at C-1 on the hapten

308

would lead to a class specific antibody. In this present investigation, the hydroxyl

309

group at C-1 on R-(–)-sal-hapten was exposed, this would benefit to the antibody

310

recognition to these compounds with hydroxyl groups (e.g. salbutamol (1), colterol (7)

311

and carbuterol (28)) and amino groups (e.g. clenbuterol (36), brombuterol (42) and

312

cimbuterol (47)) at C-1. However, the spacer of sal-hapten-4 was derived at C-1, the

313

raised antibody exhibited a poor recognition to clenbuterol (CR < 1%),36 because the

314

hydroxyl at C-1 on the hapten was not exposed.

315

C-6 Effect. The blue contour suggested that increasing positive electricity at C-6

316

would be favorable for the binding affinity enhancing (Figure 3c). The hydrogen of

317

phenolic hydroxyl on terbutaline (11) possessed a stronger positive charge

318

(Gasteiger-Huckel charge, 0.257) than both bromine (Gasteiger-Huckel charge, -0.107)

319

of brombuterol (42) and chlorine (Gasteiger-Huckel charge, -0.125) of clenbuterol

320

(36). Therefore, the cross-reactivity of terbutaline (CR, 8.3%) was higher than those

321

of the other two analytes (CR, 3.2% for brombuterol and 2.9% for clenbuterol).

322

Similarly, mabuterol (44) could not be recognized (CR, ND) because it contained a

323

negative charge trifluoromethyl (Gasteiger-Huckel charge, -0.237) at C-6. Since there

324

were no substituents at C-6 on R-(–)-sal-hapten and the hydrogen carried a positive

325

charge (Gasteiger-Huckel charge, 0.017), adding negative charge at C-6 on the hapten

326

would be favorable for the antibody recognition to the compounds contained negative

16


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327

charge groups at C-6.

328

There were yellow and white contours (steric and hydrophobic fields) near C-6

329

on sal-hapten-1 and 2 (Figure 3a, 3e), QSAR revealed that the bulky and hydrophobic

330

groups near C-6 would decrease the binding affinity, and thus be beneficial to

331

generate narrow specificity antibody. Although the hydrogen on methoxyl group of

332

DIM (13) possessed stronger positive electricity (Gasteiger-Huckel charge, 0.059)

333

than brombuterol and clenbuterol, DIM still showed a lower binding affinity (CR,

334

1.3%). This could be ascribed that the influence of steric field at C-6 was stronger

335

than the electric field.

336

The hapten spacers of sal-hapten-1 and sal-hapten-2 were derived at C-6.

337

CoMFA analysis revealed that the electrostatic field near C-6 could play an important

338

role on antibody specificity. The negative charge nitrogen of sal-hapten-2 spacer could

339

enhance the recognition of the raised antibody to the negative charge groups at C-6,

340

such as the chlorine on clenbuterol, and thus the cross-reactivity of clenbuterol (CR,

341

107%) became higher than that of salbutamol (CR, 100%) and terbutaline (CR, 10%).

342

It could be inferred that the raised antibody from sal-hapten-2 might recognize those

343

compounds with negative charge groups at C-6, but the recognition to those

344

compounds with positive charge at C-6 might decline. Therefore, the electric charge

345

of the hapten spacer at C-6 could affect the specificity spectrum of the resultant

346

antibody.

347

Sal-hapten-1 spacer contained only one carbon between the benzene ring of

348

salbutamol and the carrier protein (BSA) at C-6. This spacer and the carrier protein

17



349

could increase the steric hindrance, and then hinder the exposure of the hydroxyl

350

hydrogen at C-1 to the blue contour between C-1 and C-6. Therefore, the recognition

351

of the raised antibody to the compounds with hydroxyl or amino groups at C-1 would

352

decrease. This may be the reason that the obtained antibody against sal-hapten-1

353

could not recognize clenbuterol (CR < 0.1%).

354

C-2 Effect. The magenta, yellow and green contours were around C-2 (Figure 3a,

355

3e), this suggested that the steric field would exert effect together with the

356

hydrophobic field. The steric field of the methyl (3D-SAL (15)), the hydrogen

357

(t-butylnorsynephrine (16)) and the hydroxymethyl (R-(–)-salbutamol (3)) at C-2 was

358

weak (Figure 1). However, the methyl in 3D-SAL (ClogP, 1.598) and the hydrogen of

359

t-butylnorsynephrine (ClogP, 1.149) showed stronger hydrophobicity than the

360

hydroxymethyl of R-(–)-salbutamol (ClogP, 0.061). The cross-reactivity of 3D-SAL

361

(CR, 2285.7%) and t-butylnorsynephrine (CR, 571.4%) was higher than that of

362

R-(–)-salbutamol (CR, 100%), this suggested that the hydrophobic field on the three

363

compounds above played a dominant role in antibody binding.

364

Although bitolterol (23) contained a strong hydrophobic benzene ring at C-1 and

365

C-2 (ClogP, 5.590), the yellow contour far away from C-2 also induced a bulky steric

366

field. Since the effect of the hydrophobic was opposite to that of the steric field, and

367

the cross-reactivity of bitolterol was very low (CR, 2.6%), it could be inferred that the

368

steric field effect was stronger than its hydrophobic field effect at C-2 on bitolterol.

369

Compared with the phenolic hydroxyl group of colterol (7) at C-2 (ClogP, 0.552),

370

although the hydroxymethyl group of R-(–)-salbutamol (3) decreased the

18


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371

hydrophobicity (ClogP, 0.061), R-(–)-salbutamol (CR, 100%) still showed a higher

372

cross-reactivity than that of colterol (CR, 0.2%). Because QSAR analysis had

373

revealed that the steric field could exert a positive effect to the binding affinity

374

enhancement, it could be induced that the steric field at C-2 on colterol exerted a

375

stronger influence on the specificity more than the hydrophobic field.

376

The green and magenta contours were near C-2 on R-(–)-salbutamol (Figure 3b,

377

3e), this suggested that the optimal spacer needed strong steric and hydrophobic fields

378

near C-2. The three carbons spacer of R-(–)-sal-hapten was a suitable sized steric field

379

for the antibody generation with a class specificity. In this present investigation the

380

succinate spacer of R-(–)-sal-hapten contained a hydrophobic ester group at C-2, and

381

this hydrophobic group would increase the binding affinity. Therefore, it was

382

reasonable that the obtained antibody demonstrated recognition up to 31 β-agonist

383

compounds.

384

Chiral effect. S-(+)-salbutamol and R-(–)-salbutamol showed difference in the

385

orientation of the hydroxyl connected to chiral carbon at C-1´ (Figure 3g, 3h). The

386

hydroxyl was a hydrophilic group, it would decrease the hydrophobicity of the region

387

if it was inserted into the hydrophobic cavity (magenta contours) at C-2´ of

388

salbutamol. This was the reason that the cross reactivity of S-(+)-salbutamol (CR,

389

1.7%) and S-(+)-TER (CR, 0.4%) were much lower than R-(–)-salbutamol (CR, 100%)

390

and R-(–)-TER (CR, 4.7%).

391

It was known that ractopamine, another usual banned β-agonist in China,

392

contained a similar chiral carbon like salbutamol at C-1´. In the previous investigation

19



393

on antibody binding of ractopamine stereoisomer, the (1R,3R)-ractopamine also

394

exhibited much higher cross-reactivity (CR, 489%) than (1S,3R)-ractopamine (CR,

395

134%).37 This indicated that levo-isomer ractopamine could demonstrate a stronger

396

class specificity. Similarly, comparing the structure between R-(–)-salbutamol (3) and

397

salbutamon (5), the only difference was that the latter contained a carbonyl group

398

instead of the chiral hydrophilic hydroxyl group at C-1´of the former. However, this

399

slight structure change made an extremely difference in activity, salbutamon could not

400

be recognized by the obtained antibody (CR, ND) in the present investigation,

401

compared to the significant high cross reactivity (CR, 100%) for R-(-)-salbutamol.

402

Therefore, it could be induced that the chirality characteristic at C-1´was essential for

403

the antibody binding to β-agonist compounds.

404

Sample analysis. To confirm the accuracy of the class specific immunoassay for

405

samples, swine urine was chosen and spiked with β-agonists (salbutamol, clenbuterol,

406

brombuterol and cimbuterol as the model drugs) for the recovery evaluation. When

407

the urine was diluted 5-fold with PBST, the IC50 for salbutamol in 5-fold diluted urine

408

was close to that for salbutamol in PBST (Figure S4). Therefore, swine urine sample

409

was diluted 5-fold with PBST to eliminate the matrix interference for the further

410

ELISA analysis. The recovery for the four β-agonists ranged from 81.8% to 118.3%,

411

the coefficient of variation (CV) was below 15% (Table 2), this could meet the

412

requirement of the U.S. Environmental Protection Agency (EPA) on recovery.38

413

To confirm the reliability of the proposed ciELISA, all spiked urine samples

414

were also measured by HPLC-MS. It is found that the retention times for salbutamol,

20


Page 20 of 33

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415

clenbuterol, brombuterol and cimbuterol were at 2.0, 5.6, 6.5 and 2.9 min by

416

HPLC-MS (Figure S5). The correlation coefficients R2 were all higher than 0.96

417

(Table 2), and this confirmed that the results from ELISA had a good agreement with

418

that from HPLC-MS.

419 420

CONCLUSION

421

In summary, using an R-(–)-salbutamol derivative as the chiral immunizing

422

hapten, a class specific antibody to β-agonists and analogue was obtained. The QSAR

423

analysis revealed that strong steric and hydrophobic fields near the hapten spacer site

424

C-2 as well as a chirality at C-1´, was crucial to the class specificity of the raised

425

antibody to β-agonist compounds. However, once the spacer was linked to sites C-1 or

426

C-2´ of a salbutamol hapten, only a narrow specific antibody would be raised. And the

427

spacer charge at C-6 could affect the specificity spectrum of the resultant antibody. A

428

ciELISA was successfully established for sensitive recognition of 31 β-agonists and

429

possible analogues for the first time. With a good agreement to HPLC-MS method,

430

the proposed ciELISA was proved to be reliable for the rapid surveillance screening

431

assay of β-agonists in urine sample. This investigation will also be beneficial to better

432

understanding the class specificity mechanism and to rationally design and control the

433

immunoassay development.

434 435 436

ASSOCIATED CONTENT Supporting Information. Experimental section of hapten-protein conjugates

21



Page 22 of 33

437

preparation, antibody production, ciELISA method and molecular modeling. Figure of

438

experimental and calculated ECD spectra of R-(–)-sal-hapten. Description of the

439

identification of hapten-protein conjugates and reduced SDS-PAGE of purified

440

R-(–)-salbutamol antibody, calibration curves of matrix effects in urine sample,

441

HPLC-MS of 4 β-agonists, summary of calculated parameters of 3D QSAR models

442

and the activity values of training and test set molecules.

443 444

AUTHOR INFORMALTION

445

Corresponding author

446

*Email for Lei H. : [email protected]. Phone: +8620-8528-3925. Fax:

447

+8620-8528-0270.

448

Notes

449

The authors declare no competing financial interest.

450 451

ACKNOWLEDGEMENTS

452

This work was supported by the National Key Research and Development

453

Program of China (SQ2017YFC160089, 2016YFE0106000), Natural Science

454

Foundation

455

S2013030013338), Guangdong and Guangzhou Planned Program in Science and

456

Technology (2016201604030004, 2017B020207010), Russian Science Foundation

457

(14-16-00149).

of

China

and

Guangdong

(31701703,

458

22


31601555,

U1301214,

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533

Table 1 Specificity comparison of antibodies raised from various derived salbutamol

534

hapten

Molecules 3D-SAL t-butylnorsynephrine R-(–)-salbutamol carbuterol Rac-salbutamol DAH clenpenterol clenhexerol terbutaline salbutamol methyl ether R-(–)-TER cimbuterol bromoclenbuterol brombuterol clenbuterol bitolterol mapenterol S-(+)-salbutamol tulobuterol pirbuterol DIM pindolol S-(+)-TER HYD colterol 5-hydroxy salbutamol cimaterol salmeterol penbutolol orciprenaline propafenone arformoterol alprenolol ractopamine mabuterol clorprenaline

R-(–)-sal-hapten (this investigation) IC50 (nmol L-1) 0.07 2285.7 0.3 571.4 1.6 100 4.4 36.4 5.1 31.4 6.8 23.5 12.5 12.8 16.5 9.7 19.3 8.3 27.3 5.8 33.5 4.7 36.4 4.4 38.9 4.1 50.5 3.2 54.5 2.9 60.5 2.6 84.8 1.9 91.7 1.7 94.0 1.7 116.3 1.4 123.4 1.3 301.3 0.5 436.7 0.4 551.7 0.3 643.6 0.2 905.7 0.1 1178.5 0.1 1258.4 0.1 1464.7 0.1 2494.8 0.1 3111.2 0.1 3449.4

Four Hapten Spacer Sites Modulating Class Specificity: Nondirectional

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