High specificity detection of Pb^2+ ions by p-SCN-Bz-DTPA immunogen and p-NH2-Bn-DTPA coating antigen
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 bifunc...
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Published in | 中国环境科学与工程前沿:英文版 no. 5; pp. 729 - 736 |
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Main Author | |
Format | Journal Article |
Language | English |
Published |
2014
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Subjects | |
Online Access | Get full text |
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Summary: | 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%. |
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Bibliography: | 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%. p-SCN-Bz-DTPA, p-NH2-Bn-DTPA, leadion, polyclonal antibody 10-1013/X |
ISSN: | 2095-2201 2095-221X |