靶向造影剂(Gd-DTPA-Granzyme B)的制备

Preparation of a novel targeted MR contrast agent Gd-DTPA-Granzyme B monoclonal antibody

目的:探讨以颗粒酶B为靶向的磁共振造影剂(gadolinium-diethylenetriaminepentaacetic-granzyme B monoclonal antibody,Gd-DTPA-GB mAb)的制备方法及反应条件。方法:采用颗粒酶B单抗制成颗粒酶B靶向造影剂Gd-DTPA-GB mAb;通过基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption ionisation time-of-flight mass spectrometry,MALDI-TOF-MS)计算其相关参数,采用MTT法检测其细胞毒性,以及免疫组化法判定其免疫活性。结果:MALDI-TOF-MS鉴定结果显示:颗粒酶B单抗的质荷比高峰主要出现在133986.41处,连接Gd-DTPA后高峰主要在139736.06的位置;根据质谱结果计算得每分子抗体可以连接20个左右的Gd3+;MTT法结果显示:Gd、DTPA、Gd-DTPA,以及Gd-DTPA-GB mAb组在浓度逐渐递增的情况下对于细胞活性没有明显影响,且4组间比较差异无统计学意义(P>0.05);免疫组化结果显示:靶向造影剂(1∶175)与阳性对照组(1∶150)比较,具有较高的免疫活性。结论:在体外,本实验室设定的条件能够合成磁共振靶向造影剂Gd-DTPA-GB mAb,而且该造影剂具有良好的免疫活性,无明显细胞毒性。

Objective： To prepare a novel MRI targeted contrast agent Gd-DTPA-Granzyme B monoclonal antibody（mAb） and to test its reaction conditions.Methods： The Granzyme B mAb was coupled with DTPA,and then conjugated with Gd.The Gd-DTPA antibody was characterized using MALDI-TOF-MS.Cytotoxicity test was performed with MTT assay,and immune activation was examined with immunohistochemistry.Results： MALDI-TOF-MS demonstrated that the molecular weight shifted from granzyme B mAb（133986） to Gd-DTPA-GB mAb（139736）,which indicated the conjugation of the antibody with Gd-DTPA.The molar ratio of Gd per IgG molecule was about 20.MTT assay showed that Gd,DTPA,Gd-DTPA and Gd-DTPA-GB mAb groups did not make an impact on cell viability,and there were no significant differences among 4 groups（P〉0.05）.Immunohistochemistry results showed that compared with the positive control group the targeted contrast agent had a high immune activity.Conclusion： The novel contrast agent Gd-DTPA-Granzyme B mAb prepared in this study keeps a good immune activity and has no significant cytotoxicity.