摘要
以双功能螯合剂异硫氰酸苄基乙二胺四乙酸(ITCBE)螯合铅离子,制备得半抗原Pb-ITCBE,然后再分别与载体蛋白KLH或BSA偶联制备得免疫原Pb-ITCBE-KLH与包被抗原Pb-ITCBE-BSA,ITCBE-BSA。用二喹啉甲酸法测3种抗原的浓度,分析半抗原、抗原与载体蛋白的紫外吸收光谱,利用SDS-PAGE对3种抗原的分子量进行鉴定,用三硝基苯磺酸法检测3种抗原中的赖氨酸残基的ε-NH2被半抗原替换的程度,用石墨炉原子分光吸收法检测抗原中铅的含量。研究结果表明,免疫原与包被抗原制备成功,Pb-ITCBE-KLH、Pb-ITCBE-BSA、ITCBE-BSA的浓度依次为6.47±0.08mg/ml,6.68±0.06mg/ml,5.57±0.05mg/ml;抗原与载体蛋白的紫外吸收光谱的特征各不相同;SDS-PAGE的结果显示3种抗原的分子量均不同于各自的载体蛋白;抗原中载体蛋白ε-氨基的替换程度依次为1.86±0.74%、55.53±1.13%、54.19±1.34%;铅的含量依次为15.64±0.11μg/ml,17.33±0.15μg/ml,0μg/ml。
In this paper, isothiocyanobenzylethyl enediamine tetraacetic acid （ITCBE） was used to chelate lead ion as a bifunctional chelator for preparing artificial hapten Pb-ITCBE. The antigens which included immunogen and coating antigen were prepared through the coupling the hapten with carrier protein keyhole limpet hemocyanin （KLH） and bovine serum albumin （ BSA）, respectively. The hapten, antigens and carrier protein were scanned with ultraviolet spectrophotometer, the molecular weight of the antigens was verified with SDS-PAGE,the degree of antigen substitution was quantified using the 2,4,6-trinitrobenzene sulfonic acid （TNBS） method, and the concentration of cadmium ion in the antigen was quantified with graphite furnace atomic absorption spectrometry （GFAAS）. The results indicated the antigens were prepared successfully. The ultraviolet absorption spectrum of antigens was different with their corresponding carrier protein. The results of SDS-PAGE indicated the molecular weight of antigens was different with corresponding carrier protein. The concentration of Pb-ITCBE-KLH, Pb-ITCBE-BSA and ITCBE-BSA was 6.47 ± 0.08 mg/ml,6.68 ± 0.06 mg/ml and 5. 57 ±0.05 mg/ml. The ratio of cadmium to carder protein in antigens was 31.86 ± 1.33% ,55.53 ±1.28% and 54.19 ± 1.69% , respectively. The content of lead in the antigens was 15.64 ± 0.11 μg/ml, 17.33 ± 0.15 μg/ml and 0 μg/ml,respectively.
出处
《生物技术通报》
CAS
CSCD
北大核心
2009年第11期158-162,共5页
Biotechnology Bulletin
基金
仲恺农业工程学院自然科学基金资助项目(2009G3091416)
广东省科技计划项目(2003C20409)
国家质检总局科技项目(2004IK062)
关键词
铅
双功能螯合剂
抗原
制备
鉴定
Lead Bifunctional chelator Antigen Preparation Identification
