摘要
目的制备抗荧光素单克隆抗体免疫磁珠,并进行初步应用。方法采用B淋巴细胞杂交瘤技术制备抗荧光素单克隆抗体;制备腹水,经50%硫酸铵粗提后,再经阴离子交换树脂DE52进一步纯化单克隆抗体;采用氧化共沉淀法制备纳米磁性粒子;乳液聚合法制备羧基磁性微球;用碳二亚胺将抗荧光素单克隆抗体共价偶联于磁性微球表面,制备抗荧光素单克隆抗体免疫磁珠。用制备的免疫磁珠检测乙肝表面抗原,并与市售ELISA试剂盒的检测结果进行比较。结果共获得5株杂交瘤细胞株,其中1F12细胞株分泌的抗体效价、相对亲和力较高,特异性较好;纯化的1F12株单抗的纯度达95%,蛋白含量为2.4 mg/ml,ELISA效价为106,相对亲和力为0.2 mg/L,与FITC标记的BSA可特异性结合;纳米磁性粒子的平均粒径为150 nm,铁含量为71.63%;羧基磁性微球的平均粒径为210 nm,羧基含量约为2.15 mmol/g;每克羧基磁性微球可结合抗荧光素单克隆抗体约12 mg,制备的免疫磁珠可有效结合荧光素标记的蛋白。应用抗荧光素单克隆抗体免疫磁珠检测乙型肝炎表面抗原的灵敏度高于ELISA试剂,检测限达0.1 ng/ml。结论成功制备了抗荧光素单克隆抗体免疫磁珠,其具有应用于免疫检测分析的价值。
Objective To prepare and preliminarily apply the immunomagnetic beads coated with monoclonal antibody （McAb） against FITC. Methods McAb against FITC was prepared by B lymphocyte hybridoma technique. The prepared ascites were preliminarily extracted with 50% ammonium sulphate and further purified with anion exchange resin DE52 to obtain McAb. Nano magnetic particles were prepared by oxidation co-precipitation method, while the magnetic micro- sphere with carboxyl group by emulsion polymerization method. McAb against FITC was linked onto the surface of magnetic mierospheres with carbodiimide （EDAC） to prepare immunogmagnetic beads. Results Five hybridoma cell strains were obtained, of which 1F12 secreted the McAb with high titer, relative affinity and specificity. The purified McAb 1F12 reached a purity of 95%, with a protein content of 2. 4 mg/ml, an ELISA titer of 106, a relative affinity of 0. 2 mg/L, and showed specific binding to FITC-labeled BSA. The mean diameter and ferric content of prepared nano magnetic par- ticles were 150 nm and 71.63% respectively. However, the mean diameter and carboxyl group content of prepared magnetic microspheres with carboxy group were 210 nm and 2. 15 mmol/g respectively. Each gram of magnetic microspheres with carboxyl group was bound to about 12 mg of McAb against FITC, and the prepared immunomagnetic beads was bound to FITC-labeled protein effectively. The prepared immunomagnetic beads coated with McAb against FITC showed higher sensitivity than ELISA in determination of HBsAg, of which the detection limit was 0. 1 ng / ml. Conclusion Immunomagnetic beads coated with McAb against FITC were prepared successfully, which might be used for immunoassay.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第3期392-396,共5页
Chinese Journal of Biologicals
关键词
荧光素
单克隆抗体
免疫磁珠
Fluorescein
Monoclonal antibody
Immunomagnetic beads
