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Frontiers of Agricultural Science and Engineering

ISSN 2095-7505

ISSN 2095-977X(Online)

CN 10-1204/S

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Front. Agr. Sci. Eng.    2017, Vol. 4 Issue (3) : 366-372    https://doi.org/10.15302/J-FASE-2017132
RESEARCH ARTICLE
Synthesis of haptens and production of antibodies to bisphenol A
Xiya ZHANG1, Xiaoyun DONG2, Sijun ZHAO3, Yuebin KE4, Kai WEN1,2, Suxia ZHANG1,5, Zhanhui WANG1,2,5, Jianzhong SHEN1,2,5()
1. Beijing Advanced Innovation Center for Food Nutrition and Human Health/College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
2. Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing 100193, China
3. Department for Safety Supervision of Animal Products, China Animal Health and Epidemiology Center, Qingdao 266032, China
4. Department of Genetic Toxicology, Shenzhen Center for Disease Control and Prevention, Shenzhen 518020, China
5. Beijing Laboratory for Food Quality and Safety/National Reference Laboratory for Veterinary Drug Residues, Beijing 100193, China
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Abstract

Three immunizing haptens of bisphenol A (BPA), including two new haptens, were used to produce highly sensitive and specific polyclonal antibodies. The spacer arms of haptens for coupling to the protein carrier were located at different positions in BPA, and different length spacer arms were tested. Highly sensitive polyclonal antibodies were obtained and characterized using indirect competitive enzyme-linked immunosorbent assay (icELISA). Under optimized conditions, the half maximal inhibitory concentration (IC50) value of the best polyclonal antibody was 2.1 mg·L−1, based on coating heterogeneous antigens, and this optimal polyclonal antibody was highly sensitive toward BPA and displayed negligible cross-reactivity with bisphenol B and bisphenol E. A sensitive icELISA method utilizing the polyclonal antibody was developed for the determination of BPA in milk. In spiked samples (5, 10 and 20 mg·L−1), the recovery ranged from 80% to 102% with a coefficient of variation (CV) value below 15.8%. The limit of detection of icELISA was 1.95 mg·L−1. These results indicate that the icELISA method is suitable for the detection of BPA in milk.
Keywords bisphenol A      cross-reactivity      hapten      indirect competitive ELISA      polyclonal antibody     
Corresponding Author(s): Jianzhong SHEN   
Just Accepted Date: 20 January 2017   Online First Date: 17 February 2017    Issue Date: 12 September 2017
 Cite this article:   
Xiya ZHANG,Xiaoyun DONG,Sijun ZHAO, et al. Synthesis of haptens and production of antibodies to bisphenol A[J]. Front. Agr. Sci. Eng. , 2017, 4(3): 366-372.
 URL:  
https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2017132
https://academic.hep.com.cn/fase/EN/Y2017/V4/I3/366
Fig.1  Haptens from the literature (a) and the synthesis of haptens and antigens (b)
CodeOYPPP-BSA/OVAHHPBA-BSA/OVABHPVA-BSA/OVA
OD450IC50/(ng·mL-1)OD450IC50/(ng·mL-1)OD450IC50/(ng·mL-1)
11.25137.4––1.64336.3
21.86738.21.48745.51.58940.2
31.94823.11.248126.2––
Tab.1  Optical density (OD450) and IC50 of polyclonal antibodiesa
Fig.2  Standard curves. (a) The comparison of homogeneous and heterogeneous icELISA; (b) the standard curve of BPA in PBS and milk. n = 3.
FactorB0 (OD450)IC50/(ng·mL-1)
Coating buffer0.01 mol·L-1 PBS, pH 7.41.7966.4
0.05 mol·L-1 CB, pH 9.62.0583.1
Temperature/℃41.9895.6
252.0125.4
372.1035.2
pH6.01.5843.2
7.42.0832.1
8.02.3072.7
NaCl concentration/( mol·L-1)0.11.9802.2
0.21.6822.4
0.41.4892.9
Tab.2  Effects of various factors on the sensitivity of the icELISA
CompoundIC50/(ng·mL-1)CR/%
BPA2.1100
Bisphenol B27.57.6
BHPVA12.716.5
Bisphenol E80.72.6
17b-estradiol>1000<0.2
Diethylstilbestrol>1000<0.1
Nonylphenol>1000<0.1
Tab.3  The IC50 values and cross-reactivity (CR) of the polyclonal antibody
Antibody nameHaptenIC50/(mg·L-1)
Polyclonal antibody (this study)BPA-AMA2.1
Polyclonal antibody[29]BPA-CME1.25
Polyclonal antibody[16]BPA-CME5.4
Monoclonal antibody BBA-2187[20]BPA-CPE0.59
Monoclonal antibody[30]BHPVA2.2
Monoclonal antibody[31]BHPVA140
Polyclonal antibody[18]BHPVA200
Polyclonal antibody[1]BHPVA1100
Polyclonal antibody[13]BPA-GA1.2
Monoclonal antibody BPAB-11[15]BPA-GA230
Chicken immunoglobulins[12]BPA-GA570
Tab.4  Comparison of IC50 values reported in the literature and in this study
Added/(ng·mL-1)Observed/(ng·mL-1)Recovery/%CV/%
55.1±0.75102.0±15.015.8
108.0±0.1280.0±11.213.0
2016.7±0.1983.5±9.59.8
Tab.5  Recoveries and coefficient of variation (CV) values for BPA in milk by the icELISA (n = 3)
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