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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%.
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Keywords
p-SCN-Bz-DTPA
p-NH2-Bn-DTPA
lead ion
polyclonal antibody
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Corresponding Author(s):
Ruozhen YU
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Issue Date: 20 June 2014
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