重金属镉人工抗原的制备与鉴定

Preparation and identification of artificial antigen for heavy metal cadmium

中文关键词:  镉  人工抗原  理化性质  免疫原性

英文关键词:cadmium  artificial antigen  physicochemical properties  immunogenicity

中文摘要:

      目的 制备重金属镉人工抗原并对其进行理化性质和免疫原性鉴定。方法 以乙二胺四乙酸二钠(ethylenediaminetetraacetic acid disodium salt, EDTA·2Na)或二乙基三胺五乙酸(diethylenetriaminepentaacetic acid, DTPA)为双功能螯合剂, 将牛血清白蛋白(bovine albumin, BSA)和Cd2+进行螯合, 制备镉人工抗原, 通过二喹啉甲酸(bicinchoninic acid, BCA)法、紫外全波长扫描、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecylsulfate- polyacrylamide gel electrophoresis, SDS-PAGE)、火焰原子吸收法及动物免疫对人工抗原进行分析鉴定。结果 紫外全波长扫描和SDS-PAGE初步证明人工抗原合成成功; 人工抗原BSA-EDTA-Cd2+和BSA-DTPA-Cd2+的蛋白质质量浓度分别为0.9488、0.5506 mg/mL, Cd2+质量浓度分别为22.8、20.1 μg/mL, 说明人工抗原合成成功, 且BSA-EDTA-Cd2+和BSA-DTPA-Cd2+的偶联比分别为14.2:1和21.6:1; 用BSA-EDTA-Cd2+和BSA-DTPA-Cd2+分别免疫小鼠, 四免后血清效价均能达到1:25600, 且两组血清半数抑制浓度(median inhibition concentration, IC50)分别达到208.88和83.91 ng, 与Ba2+、Fe3+、Zn2+、Pb2+几乎没有交叉反应。结论 人工抗原BSA-EDTA-Cd2+、BSA-DTPA-Cd2+合成成功, 为制备单克隆抗体和建立重金属镉快速免疫分析技术提供理论基础。

英文摘要:

      Objective To preparethe artificial antigen of heavy metal cadmium (Cd), and identify its physical and chemical properties and immunogenicity. Methods With ethylenediaminetetraacetic acid disodium salt (EDTA·2Na) and diethylenetriaminepentaacetic acid (DTPA) as the bifunctional chelating agents, bovine serum albumin (BSA) and Cd2+ were chelated to prepare artificial antigen of cadmium, the artificial antigens were identified by bybicinchoninic acid (BCA) method, ultraviolet full-wavelength scanning, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), flame atomic absorption spectrometry and animal immunity. Results Ultraviolet full-wavelength scanning and SDS-PAGE preliminarily proved that the antigen synthesis was successful; the protein massconcentrations of artificial antigens BSA-EDTA-Cd2+ and BSA-DTPA-Cd2+ were 0.9488 and 0.5506 mg/mL, and the Cd2+ mass concentrations were 22.8 and 20.1 μg/mL, respectively; and the coupling ratios of BSA-EDTA-Cd2+ and BSA-DTPA-Cd2+were 14.2:1 and 21.6:1. Mice were immunized with BSA-EDTA-Cd2+ and BSA-DTPA-Cd2+, respectively. The serum titers of artificial antigen reached 1:25600 after 4 immunizations, and the median inhibition concentration (IC50) of serum in the 2 groups reached 208.88 and 83.91 ng, respectively, and there was almost no cross reaction with Ba2+, Fe3+, Zn2+ and Pb2+. Conclusion The artificial antigens BSA-EDTA-Cd2+ and BSA-DTPA-Cd2+ have been successfully synthesized, which provide a theoretical basis for the preparation of monoclonal antibodies and the establishment of rapid immunoassay for heavy metal cadmium.