| 65% of Chinese people eat rice as an absolutely staple food.In 2002, the Ministry of Agriculture, Rice and Product Quality Supervision and Inspection Center had sampled rice for safety testing in the national market, the results showed that the content of cadmium in rice exceeded the rate of 10.3%. In the future, Chinese agricultural product safety issues:cadmium and other heavy metal pollution,would replace pesticides to become accident-prone areas. Traditional detecting methods can not meet the demands, it’s urgent to find new, rapid, sensitive and efficient detecting methods. In this paper, we used cadmium(Cd2+) as the research object, based on the preparation of monoclonal antibody against Cd2+, to prepare colloidal gold immunochromatography strip, for establishing the on-sited and rapid detection of cadmium by utilizing immunological techniques.Macromolecular bifunctional chelator l-(4-isothiocyanate benzyl) ethylene diamine-N,N,N’,N’-tetraacetic acid (isothiocyano benzy-EDTA, iEDTA) was used to chelate Cd2+ to form six tooth complexes, then conjugate carrier ovalbumin (OVA) and bovine serum albumin (BSA), to synthesize artificial immune-antigen Cd-iEDTA-OVA and detecting-antigen Cd-iEDTA-BSA. Contents of Cd2+ in Cd-iEDTA-OVA and Cd-iEDTA-BSA,measured by graphite furnace atomic absorption spectrophoto-meter, were 174.6230μg·L-1 and 48.1881μg·L-1; the results of ultraviolet spectro-photometric scanning showed complete antigen had absorption characteristics of the carrier protein and hapten at the same timeand the content of OVA and BSA were 1.7892 mg·ml-1 and 1.8065 mg·mL-1. All the above illustrated the success of synthesizing cadmium complete antigen, and it can be used in subsequent research.Cd-iEDTA-OVA was injected into small amount of BALB/c mice at multiple sites, and antiserum was obtained. Then through cell fusion, hybridoma and cloning techniques, two specific hybridomas i.e 7F4 and 7E8 were gained. And by using octylic acid-ammonium sulfate salting out, including ascites induced in vivo, differential centrifugation and purification techniques, five monoclonal antibodies (mAb) i.e 7E8E5,7E8G9,7F4B8,7F4D6 and 7F4H8 were accessed. The result of enzyme-linked immune sorbent assay (ELISA) showed that the five mAbs were all IgG1 subtype with a high specific affinity up to 108, and could not be interfered by other ions except Hg2+. The minimum detection limit of the ELISA method established for detecting Cd2+ was 260 ng·mL-1, and the result was strictly linear with respect to the content of 260～5 OOOng·mL-1. Meanwhile, the results of SDS-PAGE electrophoresis indicated that those mAbs showed a high purity which contain only two clear bands (a heavy band and a light band). Among five mAbs,7E8E5 and 7E8G9 had relatively high titer:1:512 000 and 256 000, and high affinity:4.55×108 1·moL-1 and 2.20×1081·moL--1 as well, so we can select the better of the two to prepare colloidal gold immunochromatography strip.Trisodium citrate reduction method was introduced to prepare 13nm gold particle s, then by eyeballing, visible-ultraviolet spectrophotometry (UV) and transmitssion electron microscopy(TEM), we identified gold particles present spherical and uniform size, and it can be used to mark 7E8G9. After optimization:the optimum marked pH was 8.2, the minimum dosage for each 1mL colloidal gold was 6.0μg mAb, T-line coated was mg·mL-1, C-line coated was 1.5 mg·mL-1, and the conditions for coated and dried was at 37℃ for 60min, the best dilution of gold-labeled mAb was 0.75 times, the optimal closure temperature and time was at 25℃ for 60min, optimum ion concentration was 0.01 mol·L-1.The complete antigen Cd-iEDTA-BSA and goat anti-mouse secondary antibody were coated on a nitrocellulose membrane as the T line and C line, respectively. 7E8G9 monoclonal antibody labeled gold nanoparticles was coated on the gold standard mat. Colloidal gold immunochromatography strip, with a sensitivity of 0.2 mg·kg-1, was successfully assembled, which had a favorable repeatability, however, strong cross with Hg2+, with the highest coexist concentration of no more than 1.0 mg·kg-1; less stronger cross with Zn2+, with the highest coexist concentration of no more than 10 mg·kg-1; and almost no cross with Cu2+, Fe2+, Ca2+, Mg22++, Al3+ and Pb2+. Meanwhile the storage period was about one year. When using test strip to detect remained cadmium in rice, we can see the results had a good accordance with GFAAS. |
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