High specificity detection of Pb²⁺ ions by p-SCN-Bz-DTPA immunogen and p-NH₂-Bn-DTPA coating antigen

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%.

Graphical abstract

Keywords

p-SCN-Bz-DTPA / p-NH ₂-Bn-DTPA / lead ion / polyclonal antibody

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