论文部分内容阅读
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。
Objective: To study the 125I labeling of anti-human CD40 monoclonal antibody (mAb) 5H6 and its biological characteristics in vitro binding with ovarian cancer cell line HO8910. Methods: 125I-5H6 125I-5H6 was labeled by chloramine-T method. The labeling rate and radiochemical purity of 125I-5H6 were determined by paper chromatography. The stability of 125I-5H6 in vitro was analyzed by trichloroacetic acid precipitation method. Scatchard analysis, lindmo method and its improved method to calculate the 125I-5H6 immunocompetence, cell binding assay analysis of 125I-5H6 with HO8910 cells internalization and retention. Results: (1) 125I labeling rate of mAb5H6 was (85.4 ± 5.2)% and radiochemical purity was (99.2 ± 0.5)%. (2) The radiochemical purity of 125I-5H6 was (80.3 ± 4.7)% after it was stored in phosphate buffered saline at 4 ℃ for 7 days. The radiochemical purity of 125I-5H6 was (95.3 ± 0.8)% after stored at 37 ℃ for 24h. (3) The affinity of 125I-5H6 and HO8910 cells was Kd = (0.711 ± 0.06) nmol / L and the maximum number of Bmax was (2.17 ± 0.08) × 105 cells / cell. (4) 125I-5H6 immunoreactivity score (38.6 ± 5.4)%. (5) The retention rate of 2h125I-5H6 and HO8910 cells at 4 ℃ was (89.8 ± 6.0)%. The internalization rate of 2h125I-5H6 and HO8910 cells at 37 ℃ was 54.9 ± 2.6%. CONCLUSION: 125I-5H6 labeling with chloramine-T method has good labeling and radiochemical purity, good in vitro stability and high immunocompetence fraction, and 125I-5H6 has a high affinity with HO8910 cells and can be used Experiment in animals.