抗人CD40 mAb 5H6的^125I标记及其与卵巢癌细胞株HO8910体外结合的生物学特性  

Preparation of iodine-125 labeled anti-human CD40 monoclonal antibody 5H6 and its binding properties in ovary cancer cell line HO8910 in vitro

作　　者：罗先富[1] 章斌[2] 马泓冰[1] 汤琳[1] 周璇[1] 徐颖[1] 吴翼伟[2] 张学光[1] 

机构地区：[1]苏州大学医学生物技术研究所,江苏省医学临床免疫学重点实验室,江苏苏州215007 [2]苏州大学附属第一医院核医学科,江苏苏州215006

出　　处：《细胞与分子免疫学杂志》2008年第6期590-593,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30571690);国家高新技术研究发展计划(863)资助项目(2006AA02A254);教育部博士点专项基金资助项目(20050285014)

摘　　要：目的:研究抗人CD40单克隆抗体(mAb)5H6的125I标记及与卵巢癌细胞株HO8910体外结合的生物学特性。方法:氯氨-T法125I标记mAb5H6(125I-5H6),纸层析法测定125I-5H6的标记率和放射化学纯度,三氯醋酸沉淀法分析125I-5H6体外稳定性,细胞结合饱和实验进行Scatchard分析,lindmo法及其改良方法计算125I-5H6的免疫活性分数,细胞结合实验分析125I-5H6同HO8910细胞的内化和滞留。结果:(1)mAb5H6的125I标记率为(85.4±5.2)%,放射化学纯度为(99.2±0.5)%。(2)125I-5H6存放于4℃磷酸缓冲液中7d后其放射化学纯度为(80.3±4.7)%,在37℃血浆中存放24h后其放射化学纯度为(95.3±0.8)%。(3)125I-5H6与HO8910细胞亲和力Kd=(0.711±0.06)nmol/L,最大结合位点数(Bmax)=(2.17±0.08)×105个/细胞。(4)125I-5H6免疫活性分数达(38.6±5.4)%。(5)4℃2h125I-5H6同HO8910细胞滞留率为(89.8±6.0)%,37℃2h125I-5H6同HO8910细胞内化率达(54.9±2.6)%。结论:氯氨-T法进行125I-5H6标记具有良好的标记率和放射化学纯度,以及良好的体外稳定性和较高的免疫活性分数,且125I-5H6与HO8910细胞具有很高的亲和力,可用于动物体内实验。AIM： To prepare iodine-125 labeled anti-human CD40 monoclonal antibody 5H6 （^125H6） and investigate the binding properties of ^125I-5H6 to HO8910 cells in vitro. METHODS： The mAb 5H6 was labeled with Na ^125I using chloramine-T method. The labeling efficiency and radiochemical purity of ^125I-5H6 were measured by paper chromatography. The stability of ^125I-5H6 in vitro was monitored by trichloroacetic acid （TCA） precipitation. The dissociation constant （Kd） of ^125I-5H6 from HO8910 cells and the numbers of maximum binding sites （Bmax） were obtained by Scatchard analysis. Immunoreactivity of ^125I-5H6 to HO8910 cells was determined by ＂Lindmo＂ assay and its improved method. Intemalization and retention of ^125I-5H6 by HO8910 cells were studied by cell binding experiments. RESULTS. （ 1 ）The labeling efficiency of ^125I-5H6 was （ 85.4 ± 5.2 ） % and its radiochemical purity was （99.2 ± 0.5 ） % . （2） Radiochemical purity of ^125I-5H6 was （80.3 ± 4.7 ） % after 7 days in 4℃ PB （phosphate buffer） and （95.3 ±0.8）% after 24 hours in 37℃ plasma. （3）At 4℃, Kd for ^125I-5H6 to HO8910 cells was （0.711 ±0.06） nmol/L and Bmax was （2.17 ±0. 08） × 10^5 sites/cell. （4）Immunoreactivity of ^125I-5H6 was （38.6 ±5.4）%. （5） Retention rate of ^125I-5H6 with HO8910 cells was （89.8 ±6.0）% after 2 hours at 4℃ and internalization rate of ^125I-5H6 by HO8910 cells was （54.9 ± 2.6）% after 2 hours at 37℃. CONCLUSION： ^125I-5H6 possesses good labeling efficiency and perfect radiochemical purity. ^125I-5H6 has suitable stability in vitro and its immunoreactivity was not low. The high affinity of ^125I-5H6 with HO8910 cells will benefit further animal study in vivo.

关 键 词：^125I标记 氯氨-T法 CD40 卵巢肿瘤 单克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]