Development Of An Immunochromatographic Assay For The Detection Of Cadmium Ions

Cadmium was connected with a carrier protein(BSA) via a bi-functionalchelator(ITCBE) to make the complete detect antigen(Cd-ITCBE-BSA) and thecontrol detect antigen(ITCBE-BSA).The complete antigen concentration wasmeasured by BCA method；SDS-PAGE electrophoresis qualitative method, ultravioletspectrum scanning and graphite furnace atomic absorption spectroscopy(GFAAS)method were used for identifying the prepared antigen.The result of BCA illustratedthat the actual protein concentration of Cd-ITCBE-BSA were2mg/mL. Reslts fromultraviolet spectrum scanning and SDS-PAGE showed that the maximum absorbtionpeaks of BSA, ITCBE-BSA and Cd-ITCBE-BSA reduced in turn and the relationshipof their migration rate in gel was:BSA＞ITCBE-BSA＞Cd-ITCBE-BSA.The resultsof GFAAS indicated that cadmium ion concentration in Cd-ITCBE-BSA solution wasreally high, achieving28.55μg/mL,and cadmium ion in BSA solution, ITCBE-BSAsolution and buffer solution was almost zero. All the results above indicated thehapten was surely coupled into protein,and the antigen synthesis was successful.The frozen hybridoma cell line B8against cadmium which was preserved byour laboratory was recovered and cultured in large scale in an carbon dioxideincubator at37℃.Then cells were peritoneally injected to Balb/c mice which had beentreated with paraffin and incomplete Freund adjuvant, and after7d-10d, ascites washarvested.Caprylic-ammonium sulfate was used to purify the ascites preliminarily andthe HiTrap Protein G HP chromatography was applied to further purification of theascites.Then antibody IgG1was harvested.The concentration of the purified antibodydetermined by BCA method was2mg/mL and its activity characterized byindirect-ELISA was1:64000.The purity of the IgG1antibody was analyzed bySDS-PAGE, displaying only two clear bands which were heavy chain and light chain, and no other protein bands in gel.All the results could meet the demands of furtherexperiments.About20nm colloidal gold particles were prepared to label the monoclonalantibody.Based on the principle of competitive immunoassay,animmunochromatographic assay(ICA) was developed.The enrichment method usingnanometer titania was combined with ICA to detect the standard samples and thespiked samples.The results showed: the enriching times was30times and the wholeassay can be finished within50min; The lower limit of quantitation was6.7ng/mL,the test strips could be stored for8weeks at4℃and the test strips had across-reactivity only with Hg2+assessed with naked eye. Results betweenimmunochromatographic assay(ICA) and inductively coupled plasma-massspectrometry(ICP-MS) had a high coincidence which meet the national test standardof environmental water samples in agriculture(GB3838-2002)。...