Establishment of ELISA for Detecting Cd-MT of Pteria Penguin and Preliminary Study of ELISA-kit
|School||Guangdong Ocean University|
|Course||Of Food Science|
|Keywords||Pteria penguin Cd-MT polyclonal antibody indirect enzyme-linked immunization adsorption method ELISA-k|
Cadmium （Cd） is chronic accumulative toxicant, which is toxic to kidney, lung, liver, nervous system and blood system. Cd also has carcinogenic, teratogenic and mutagenic effects. Living environments are contaminated with Cd extensively Because all kinds of activities of human, especially the development of modern industrial. More and more attention is also paid to the harm to human and animals of Cd in three wastes.Metallothioneins （MT） is a kind of low molecular weight, cysteine-rich proteins, which exists widely in organism. Its synthesis can be induced by metals in the environment. When in vitro or in vivo metal content exceeds a certain concentration, its expression will be induced. Besides, the expression level relates directly to metal content concentration in the environment. Therefore, in the 1970s, some researchers suggested that the amount of MT contained in aquatic organisms could be used as biomarker of water environment pollution. Bivalves are benthic species that live in the boundary layer between water and deposition. Generally, the heavy metal contents in these organisms closely related to the pollution degree of water environment and reflect truly environment pollution, so MTs can serve as biomarkers of heavy metal pollution of water ecologic environment. In this study, Pteria penguin were induced to synthesize CdCl2 by injecting CdCl2 and Cd-MT was isolated and purified, polyclonal antibody against Cd-MT was prepared and ELISA （enzyme-linked immunization adsorption method） system for detecting Cd-MT was established, which might provide a rapid, sensitive, simple and convenient method for the detection of Cd-MT in shellfish. The results are as follows:1. Induction, purification and identification of Pteria penguin Cd-MT: Pteria penguin were induced to synthesize CdCl2 by injecting different concentrations of CdCl2, the results showed that the content of Cd-MT in Pteria penguin was increased with induced concentration of Cd2+. Sephadex G-50 gel?column chromatography and DEAE Sepharose Fast Flow ion-exchange column chromatography were used to purify the Cd-MT, SDS-PAGE showed melocular weights of purified Cd-MT and its dimer were 9kDa and 18kDa respectively. Every Cd-MT cintained 19.2 thiols. The specific absorption at 254nm of Cd-MT disappeared after demetalization and the absorption value declined.2. The preparation, purification and identification of rabit anti-Pteria penguin Cd-MT polyclonal antibody: Cd-MT and BSA were coupled and molecular weight of the conjugate was lager than 70kDa. Cd-MT-BSA conjugate was then used as antigen for immunizing of domestic rabbit and antiserum with titer as high as 1:12800 was prepared. Saturated ammonium sulfate deposit approach and DEAE-Sephadex A-50 column chromatography were used to separated and purified the antibody and electrophoretic pure polyclonal antibody with molecular weight of 35×103 was obtain. The polyclonal antibody showed strong cross-reactivity with Cd-MT of Pteria penguin, Pinctada martensii, Perna viridis, Chlamys farreri, Crassostrea rivularis, which meaned the ELISA method could be used to analysis the Cd-MT of these shellfishes.3. Establishment of indirect non-competitive ELISA for the analysis of Pteria penguin Cd-MT: The optimum conditions were determined by optimizing. Pteria penguin Cd-MT was dissolved in saline water and incubated for 2h at 37℃. 2% skim milk was added as a blocking solution and incubated for 30min at 37℃. The Cd-MT antibody （25μg/mL） was added and incubated for 1h at 37℃. HRP-labeled secondary antibody （1:4000 dilution） was added and incubated for 1h at 37℃. Color development time was 10min. The detection limit of this method was （4.563±0.051） ng/mL and the linear range of detection was from 9.75ng/mL to 2×104ng/mL. The relative standard deviation of intra-test and inter-test were no more than 11.6% and 6.7% respectively. The recoveries ranged from 83.7% to 119.0%. According to the method, Cd-MT concentrations of MT crude extracts of whole viscera organ, gill, adductor muscle, mantle and visceral from Pteria penguin were 15.21, 8.76, 38.74, 11.17, 76.04ng/g respectively, which matched well with the concentrations of cadmium.4. Establishment of indirect competitive ELISA and primarly research of ELISA-kit for the analysis of Pteria penguin: Indirect competitive ELISA was established and ELISA-kit for detecting Pteria penguin Cd-MT was made on the basement of that. Precision and accuracy tests showed that Intra-assay relative standard deviation （RSD） was less than 8.7% and inter-assay RSD less than 5.2%. The recoveries ranged from 84.4% to 132.9%, which meaned this method had a good stability and accuracy. The detection limit was 1.58ng/mL. IC50 was 405.92ng/mL and the linear range of detection ranged from 3.9ng/mL to 2×103ng/mL. The storage expriments showed that the ELISA-kit could store for 7d at 37℃and for at least two months at 4℃. The Cd-MT concentrations of Pteria penguin tissues were analyzed by the ELSIA-kit and the results were consistent with the results analyzed by indirect non-competitive ELISA.In this study, ELISA was established for detection of MT in Pteria penguin organs and ELISA-kit was preliminary studied, which provided a quick and convenient method to monitor the Cd pollution of aquatic environment.?