南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (09): 1308-.

• • 上一篇    下一篇

血管内皮生长因子受体2单抗的靶向超声造影剂制备及体外评价

刘红梅,韩小华,杨莉,纪丽景,李素淑   

  • 出版日期:2013-09-20 发布日期:2013-09-20

Detection of binding capability of targeted KDR ultrasound contrast agent in vitro for evaluating endometrial receptivity

  • Online:2013-09-20 Published:2013-09-20

摘要: 目的探讨携血管内皮生长因子受体2(KDR)单抗的靶向脂质微泡的制备方法,并对其物理特性、生物活性、靶向黏附稳定
性进行体外测定。方法采用声振仪制备生物素化脂质微泡(MB-B),再应用生物素-亲和素桥连技术构建携KDR单抗的靶向
微泡(MB-BAB-KDR)。荧光显微镜下观察MB-B、单纯单抗微泡(MB-B-KDR)、MB-BAB-KDR与荧光二抗孵育后荧光强度。
利用平行板流动腔技术体外模拟生理血流剪切力条件,评价靶向微泡的黏附效能。结果制备的靶向微泡MB-BAB-KDR呈圆
球形,粒度分布均匀。与荧光二抗孵育后,MB-BAB-KDR发出明亮的绿色荧光(Ⅱ级),而MB-B-KDR显示微弱的绿色荧光(Ⅰ
级)、MB-B无荧光(0级)。体外黏附实验显示,MB-BAB-KDR结合数目随着小鼠KDR Fc包被浓度的增高而增加(P<0.05),相
同浓度下,随着时间的增加,MB-BAB-KDR 结合数目增加。结论成功构建生物素-亲和素桥连的靶向脂质体微泡
MB-BAB-KDR,体外黏附实验显示MB-BAB-KDR具有黏附稳定性,且与时间、浓度呈正相关。

Abstract: Objective To prepare a new targeted liposome ultrasonic contrast agent with anti-KDR antibody that binds
specifically with KDR as the main receptor of VEGF and evaluate its physical characteristics, biological activity and specific
binding capability in vitro. Methods A sonicator was used to prepare the biotinylated lipid micro-bubbles (MB-B), and
biotin-avidin-mediated technique was used for attachment of anti-mouse KDR monoclonal antibody to the micro-bubble shell
to generate MB-BAB-KDR. MB-BAB-KDR was incubated with fluorescent second antibody to assess the link condition, and the
control groups were the MB-B and micro-bubbles with the antibody alone (MB-B-KDR). A parallel plate flow chamber system
was used to characterize micro-bubbles attachment efficiency to KDR Fc. Results The surface of the micro-bubbles could carry
KDR antibody through the biotin-avidin bridge and MB-BAB-KDR were spherical and well-distributed. After incubation with
the second antibody, MB-BAB-KDR could be observed to emit bright green fluorescence (Grade II) as compared with little or
weak fluorescence in the control MB-B group (Grade 0) and MB-B-KDR group (Grade I). Targeted micro-bubbles bound to
KDR Fc increased as the KDR Fc concentration increased (P<0.05). Conclusion The targeted liposome contrast agent linked
with KDR antibody by biotin-avidin bridge we prepared shows an increased binding number as the KDR Fc concentration
increases, which provides a novel approach to molecular imaging study of endometrial receptivity.