| The traditional detection methods for heavy metals such as atomic absorption spectrometry. inductively coupled plasma mass spectrometry can meet the needs of common analysis for mercury and other heavy metals, but these methods are expensive, time consuming and does not meet the need for rapid detection. Rapid, sensitive, suitable and on-site immunoassay may provide a new choice for detection of mercury and other heavy metals.This study was designed to prepare monoclonal antibodies with high affinity and specificity to mercury, and then the monoclonal antibody would be used to form fast immunoassay for mercury ion. Mercury ions were chelated with aminobenzyl-EDTA and ITCBE. respectively, and then the resulting complexes were conjugated with carrier protein to prepare antigens for mercury. The conjugates were chatacterized by SDS-PAGE. UV scanning, atomic absorption spectrometry and animal immunization. BALB/c mice were injected with KLH-aminobenzyl-EDTA-Hg that presented better immunogenicity than other conjugates. The spleen cells of the mice were fused with SP2/0, and then only the hybridoma of which supernatant react strongly to BSA-aminobenzyl-EDTA-Hg and did not react to BSA-aminobenzyl-EDTA were carried out the succedent clone. The cell line which secreted IgG antibody was selected to prepare ascites. The purified monoclonal antibodies were used to develope competitive indirect ELISA method and colloidal gold immunochromatography.The immunogen (KLH-aminobenzyl-EDTA-Hg,KLH-ITCBE-Hg). detection antigen (BSA-aminobenzyl-EDTA-Hg, BSA-ITCBE-Hg) and control detection antigen (BSA-aminobenzyl-EDTA, BSA-ITCBE) were successfully prepared with aminobenzyl-EDTA, ITCBE. The immunogen KLH-aminobenzyl-EDTA-Hg was more easy of inducing the anminal to produce sepefic antibody KLH-ITCBE-Hg. Then the immunogen KLH-aminobenzyl-EDTA-Hg was used to immune BALB/c mice to prepare monoclone antibody for mercury, and two hybridoma cell lines were obtained (3D9 and 5F7). The antibody secreted by 3D9 subclass was IgM, and 5F7 was IgG. The hybridoma (5F7) was secleted for ascites production, and ascites was purefied The purifed ascite (McAb) titer to chelated mercury was 1.28×106. and the affinity constant Ka was 4.31×109 L/mol. The cross-reactivity rate of the monoclonal antibody with Cd2-and Pb2- were 0.94% and 0.381%. respectively, and the cross-reaction rates with other metal tons were less than 0.2%. The prepared monoclonal antibody (5F7) was used to form competitive indirect ELISA (CI-ELISA) for detection of mercury ions, and the cross-reactivity rate of the method with Cd2+ and Pb2+ were 0.97% and 0.493%, respetively, and the cross-reactivity rate with other metal ions were less than 0.2%. The minimum detection limit of the CI-ELISA for mercury was 0.042μg/L, and the detection range was from 0.087 to 790.4μg/L, intraassay coefficient of variation (CV) was 4.36% and interassay coefficient of variation was 6.29%; the recovery rate for standard solution varied within 96.47% to 104.6%. The method correlated well with graphite furnace atomic absorption spectrometry for water samples detection, and the recovery rate was from 88.82% to 104.64%. Colloidal gold immunochromatographic technology for detecting mercury, which was based on monoclonal antibody (5F7) was developed. The test strip did not react with other ions in water, and the detection limit was 0.8μg/L. |
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