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| Competitive combination with uranium(Ⅵ) between chelating agent and metallothionein |
| ZHANG Xuxia, BAO Yizhong, WANG Mengmeng, CHEN Honghong |
| Department of Radiation Biology, Institute of Radiation Medicine, Fudan University, Shanghai 200032 China |
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Abstract Objective To establish a new method for the in vitro screening of U(Ⅵ) decorporation agents by exploring the competitive combination with uranium (Ⅵ) between chelating agent and metallothionein (MT). Methods Competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect the competitive ability of chelating agents to mobilize the U(Ⅵ) and Zn2+ binding to MT. MT antigen was coated on the surface of well of 96-well plates and then pretreated with U(Ⅵ) or Zn2+ and then individually treated with chelating agents of CBMIDA-CaNa2, BPCBG and DTPA-CaNa3 followed by reacted with MT antibody and the secondary antibody linked with horseradish peroxidase (HRP) enzyme. The absorption values were detected by spectrophotometry at 490 nm after the chromogenic reaction of OPD. Results The absorption values of this testing system increased with higher concentrations of coating MT and MT antibody, and the optimal concentration of coating MT and MT antibody were both 2 μg/mL. Similar to the effects of Zn2+, U(Ⅵ) treatment also reduced the absorption values of testing system, with the increase of U(Ⅵ) and Zn2+ concentration, and the optimal concentration of U(Ⅵ) and Zn2+ were both 300 μg/mL. Treatment with CBMIDA-CaNa2 and BPCBG significantly increased the absorption values of U(Ⅵ)-treated testing system, and effects of CBMIDA-CaNa2 were higher than that of BPCBG. DTPA-CaNa3 treatment had no effects on the absorption values of U(Ⅵ)-treated testing system. Importantly, the order of competitive ability of chelating agents to mobilize the U(Ⅵ) binding to MT was consistent with the results of previous in vitro cell culture and in vivo animal decorporation experiments for U(Ⅵ). In addition, treatment with the above chelating agents had no effects on the absorption values of Zn2+-treated testing system. Conclusion CBMIDA-CaNa2 and BPCBG can chelate U(Ⅵ) with MT, and the competitive chelation ability of CBMIDA-CaNa2 for U(Ⅵ) binding to MT is better than that of BPCBG while DTPA-CaNa3 has no obvious effect. This competitive ELISA assay can be used as a screening method for U(Ⅵ) decorporation agents in vitro which is simple, quick and high throughput.
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Received: 08 September 2018
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