摘要
通过双功能螯合剂S-2-(4-异硫氰苄基)-二乙烯三胺五乙酸(ρ-SCN-Bn-DTPA)将锌离子(Zn2+)分别与载体蛋白匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)和牛血清白蛋白(bovine serum albumin,BSA)偶联。通过二喹啉甲酸(bicin-choninic acid,BCA)法测抗原蛋白浓度,对抗原、KLH和BSA分别进行紫外分光光度计扫描,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)进行定性鉴定,利用石墨炉原子吸收分光光度法检测抗原中Zn2+含量等,成功获得了免疫抗原Zn-DTPA-KLH和检测抗原Zn-DTPA-BSA、DTPA-BSA。用Zn-DTPA-KLH免疫BALB/c小鼠,通过细胞融合,极限稀释法亚克隆,间接酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)筛选,获得了1株稳定分泌抗重金属锌抗体的杂交瘤细胞株(Z1A5)。Z1A5染色体数目在100以上,所分泌的抗体为IgM亚类,轻链为kappa型,腹水型抗体效价高达1∶51 200。本研究为锌离子残留免疫学检测方法的建立提供了物质及技术基础,对提高风险评估工作的效率和质量,保障食品安全有重要现实意义。
Zinc ions were coupled to protein carrier keyhole limpet hemocyanin（KLH）and bovine serum albumin（BSA）,respectively,using bifuntional chelator（S-2-（4-Isothiocyanatobenzyl）-Diethylenetriamine Pentaacetic Acid,ρ-Scn-Bn-DTPA）.After the assay for concentration of the antigen with bicinchoninic acid（BCA）method,the antigen and carrier protein（KLH and BSA）were scanned with ultraviolet spectrophotometer,and verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.Concentration of zinc ion in antigen determined with graphite-furnace atomic absorption spectrometry.The results indicated antigen Zn-DTPA-KLH,Zn-DTPA-BSA and DTPA-BSA were synthesized successfully.Monoclonal antibodies against zinc were generated by immunizing BALB/c mice with zinc conjugated antigen（Zn-DTPA-KLH）.The hybridoma cell line producing stably specific monoclonal antibodies against zinc was by produced fusion of murine splenocytes and SP2/0 myeloma cells,subcloned by the limiting dilution and screened by indirect enzyme-linked immunosorbent assay（ELISA）,named Z1A5.The chromosome numbers of Z1A5 was above 100.The type of secrete antibodies from Z1A5 was IgM,kappa.The antibody titers of ascites was up to 1∶51 200.These data laid a potency of establishing immunoassays methods of determining zinc ion residues and had the realistic significance for improving the efficiency and quality of risk assessment.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第7期214-220,共7页
Biotechnology Bulletin
基金
国家高技术研究发展计划(“863”计划)(2007AA10Z439)
国家自然科学基金项目(30671205)
北京市科委课题(Z09090501040901)
北京市农林科学院科技创新能力建设专项(KJCX201101010)
