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Frontiers of Environmental Science & Engineering

ISSN 2095-2201

ISSN 2095-221X(Online)

CN 10-1013/X

Postal Subscription Code 80-973

2018 Impact Factor: 3.883

Front. Environ. Sci. Eng.    2014, Vol. 8 Issue (5) : 729-736    https://doi.org/10.1007/s11783-013-0611-3
RESEARCH ARTICLE
High specificity detection of Pb2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen
Ruozhen YU(), Zheng FANG, Wei MENG, Zhenguang YAN, Lina DU, Hong WANG, Zhengtao LIU
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China
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Abstract

Only one bifunctional metal-chelator was used to prepare immunogen and coating antigen in all of the previous researches. However, the antibody-specific recognition to the spacer arm of the bifunctional metal-chelator might lower the specificity of heavy metal ions immunoassay. Two different bifunctional metal-chelators were adopted to prepare the immunogen and coating antigen respectively in our study to avoid this problem. The conjugates of keyhole limpet hemocyanin (KLH) and p-SCN-Bz-DTPA-Pb were used as immunogen, whereas the conjugates of bovine serum albumin (BSA) and p-NH2-Bn-DTPA-Pb were used as coating antigen. Polyclonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay (ELISA) was adopted to detect Pb2+ ion solutions prepared by Pb2+ standard solution and ultrapure water. In the mixing microplate, DTPA and Pb2+ ions formed chelates and combined with specific antibodies. After incubation, the DTPA-Pb and the antibodies complex were added into the wells of the reaction microplate. The reaction microplate was coated by the conjugates of BSA and p-NH2-DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb2+ concentration ranges from 0.01 to 100 mg·L−1. The IC50 of the indirect competitive ELISA is 0.23±0.04 mg·L−1 Pb2+ ion. The cross-reaction with Cd2+, Cu2+, Fe2+, Mn2+, Zn2+, and other divalent ions were less than 5%.

Keywords p-SCN-Bz-DTPA      p-NH2-Bn-DTPA      lead ion      polyclonal antibody     
Corresponding Author(s): Ruozhen YU   
Issue Date: 20 June 2014
 Cite this article:   
Ruozhen YU,Zheng FANG,Wei MENG, et al. High specificity detection of Pb2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen[J]. Front. Environ. Sci. Eng., 2014, 8(5): 729-736.
 URL:  
https://academic.hep.com.cn/fese/EN/10.1007/s11783-013-0611-3
https://academic.hep.com.cn/fese/EN/Y2014/V8/I5/729
Fig.1  Structural schematic illustration of conjugates of two kinds of DTPA derivatives (bifunctional chelator) and macromolecule carriers: (a) p-SCN-Bz-DTPA and KLH conjugate; (b) p-NH2-Bn-DTPA and BSA conjugate
Fig.2  MALDI-TOF-MS diagram of BSA
Fig.3  MALDI-TOF-MS diagram of BSA and p-NH2-Bn-DTPA conjugates
Fig.4  MALDI-TOF-MS diagram of BSA and p-SCN-Bz-DTPA conjugates
Fig.5  ELISA detection results of Pb2+ standard solution control testing: (a) dose–response curve of ELISA used BSA-p-NH2-Bn-DTPA-Pb2+ as coating antigen and KLH- p-NH2-Bn-DTPA-Pb2+ specific antiserum; (b) dose–response curve of ELISA used BSA-p-NH2-Bn-DTPA-Pb2+ as coating antigen and KLH- p-SCN-Bz-DTPA-Pb2+ specific antiserum
Fig.6  ELISA detection results of Pb2+ standard solution
metal ions IC50 /(mg·L−1) IC50 /(μmol·L−1) CR /%* reported in Literature (μmol·L−1)
CR /% [22]
Pb 2.89 13.95 100 100
Hg 21.70 108.18 12.89 2.49
Ca 28.37 707.87 1.97 <0.269
Mg 74.82 3078.38 0.45 <0.269
Cd 42.10 374.52 3.72 2.60
Cu 35.47 558.19 2.50 2.59
Fe 128.82 2306.71 0.60 1.19
Mn 16.86 306.89 4.54 <0.269
Zn 21.67 331.40 4.21 <0.269
Tab.1  Antiserum and CR of common divalent metal ions
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