氯胺T法标记B7-2单抗6C8的方法学研究

Investigation of labeling monoclonal antibody 6C8 of B7-2 with chloramine T

目的:建立125I标记B7-2单抗6C8的方法,探讨其最佳标记条件。方法:氯胺T法标记单抗6C8,改变标记条件:抗体的量分别为3μg、6μg、9μg、12μg;125I的活度分别为0.925MBq、1.85MBq、3.7MBq、7.4MBq、14.8MBq;CH-T的量分别为10μg、20μg、30μg、40μg、50μg;反应时间分别为30s、1min、2min、4min;每次实验均重复3次,根据实验结果调整标记条件。标记好后,找出最佳标记条件,在生理盐水和人血清中分别作不同时间的标记率稳定性测定,以30min、1h、2h、4h、6h、24h为时间点,选出标记率最高的时间点。纸层析法测定标记率及放化纯。结果:抗体量对标记率没有影响;Na125I3.7MBq时标记率为(74.53±5.12)%,放化纯为(90.40±3.01)%;CH-T20μg时标记率为(81.00±14.75)%,放化纯为(95.17±4.45)%;反应时间30s时标记率为(80.67±13.32)%,放化纯为(88.93±10.16)%。室温下放置3d标记率为(83.12±8.47)%,加入人血清后放置24h标记率为(5.23±0.90)%。结论:上述氯胺T法最佳标记条件为:Na125I3.7MBq,氯胺-T20μg,6C8抗体6μg,Na2S2O5100μg,反应时间30s。该方法具有较高的标记率和较差的稳定性。

Objective： To establish a method for labeling monoclonal antibody 6C8 of B7-2 with chloramine T and to discuss the best labeling conditions. Methods： Labeling monoclonal antibody 6C8 with chloramine T, and several labeling conditions were used for tests. The quantity of antibody was 3 μg, 6 μg, 9 μg and 12 μg, respectively; the activity of ^125I was 0.925 MBq, 1.8S MBq, 3.7 MBI, 7.4 NBq and 14.8 MBq, respectiveiy and the weight of CH-T was 10 μg, 20 μg, 30 μg, 40 μg, 50 μg and the reaction time was 30 s, 1 min, 2 min and 4 min. Each experiment repeated three times. Labeling conditions were regulated according to the experiment results until the best conditions were found. Then the stability of labeling rate was detected in PS and human serum, and the time was seleced when the labeling rate was the highest. Paper chromatography was used for measuring the labeling rate and radiochemical purity. Results： The quantity of 6C8 had no impact on labeling rate. When NaOmi was 3.7 NBq, the labeling rate and radiochomical purity was （74.53± 5.12） % and （90.40± 3.01） %, respectively. When CH-T was 20 μ g, those was （81.00 ± 14.75） % and （95.17 ± 4.45） %, respectively. When the reaction time was 30 s, the labeling rate and radiochemical purity was （80.67 ± 13.32） % and （88.93 ± 10.16） %, respectively. The rate of labeling was （83.12 ± 8.47） % when the labeling antibody in room temperature was tested 3 days later. The labeling antibody in human serum was tested after 24 hours, its＇ labelling rate was （5.23 ± 0.90） %. Conclusion： The best labeling conditions are： Na^125I 3.7MBq, CH-T 20 μg, 6C8 6 μg, Na2S2O5 100 μg and reactive time 30 s. The method has high labeling rate but low stability.