High specificity detection of Pb~(2+) ions by p-SCN-Bz-DTPA immunogen and p-NH_2-Bn-DTPA coating antigen

摘要

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 imrnunoassay. 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-NH_2-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 Pb~(2+) 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-NH_2-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 l00mg·L~(-1). The IC50 of the indirect competitive ELISA is 0.23±0.04mg·L~(-1) Pb~(2+) ion. The cross-reaction with Cd~(2+), Cu~(2+), Fe~(2+), Mn~(2+), Zn~(2+), and other divalent ions were less than 5%.