High specificity detection of Pb^2＋ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen

• Published in: 中国环境科学与工程前沿：英文版 no. 5; pp. 729 - 736
• Main Author: Ruozhen YU Zheng FANG Wei MENG Zhenguang YAN Lina DU Hong WANG Zhengtao LIU
• Format: Journal Article
• Language: English
• Published: 2014
• Subjects: 免疫原; 包被抗原; 特异性检测; 酶联免疫吸附测定; 重金属离子; 金属螯合物; 铅离子; 间接竞争ELISA
• ISSN: 2095-2201

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 sentrn albumin （BSA） and p- NH2-Bn-DTPA-Pb were used as coating antigen. Poly- clonal antibodies specific to DTPA-Pb chelates were obtained from rabbits. Indirect competitive enzyme-linked immunosorbent assay （ELISA） was adopted to detect Pb^2＋ ion solutions prepared by Pb^2＋ standard solution and ultrapure water. In the mixing microplate, DTPA and Pb2＋ ions formed chelates and combined with specific anti- bodies. 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 ofBSA andp-NH2-DTPA-Pb, which competed for the specific antibodies. The result signals presented a good sigmoid curve when the Pb^2＋ concentration ranges from 0.01 to 100mg·L^-1 The IC50 of the indirect competitive ELISA is 0.23±0.04mg·L^-1 Pb2＋ ion. The cross-reaction with Cd^2＋, Cu^2＋, Fe^2＋, Mn^2＋, Zn^2＋, and other divalent ions were less than 5%.