抗体偶联白蛋白超声造影剂的制备及体外靶向性研究

Studies on preparation and immunological characterization of albumin ultrasound contrast agents with monoclonal antibody binding

目的制备单克隆抗体修饰的白蛋白微气泡,并探讨其体外靶向性作用。方法采用N-琥珀酰亚胺基-3-(2-吡啶二硫)-丙酸酯(SPDP)交联法将鼠抗人血管细胞粘附分子(VCAM-1)单克隆抗体与白蛋白微气泡进行共价偶联,采用玻片凝集法检验抗体是否与白蛋白微气泡交联;体外培养高表达VCAM-1抗原的损伤血管内皮细胞,通过抗体修饰微气泡与该细胞的特异性结合作用检验该微气泡的靶向性作用。结果SPDP交联法制备的免疫微气泡的玻片凝集反应呈阳性;损伤内皮细胞(细胞膜表面高表达VCAM-1抗原)周围可见大量免疫微气泡聚集,而正常内皮细胞(细胞膜表面无VCAM-1抗原表达)表面未见明显免疫微气泡附着。结论应用SPDP交联法能够将抗体有效地偶联到白蛋白微气泡表面,体外实验证实了其与靶细胞的特异性结合作用。

Objective To prepare albumin microbubble with monoclonal antibody binding by N- succinimidyl 3-（2-pyridyldithio） propionate （SPDP） conjugation technology. This kind of microbubbles＇ targeting function was further explored in vitro studies. Methods The rat anti human vascular cell adhesion molecule-1 （VCAM-1） monoclonal antibodies were covalently coupled to the albumin microbubbles＇ shells by SPDP. Slide agglutination test was used to detect whether antibody biding with the microbubbles. The TNF-a activated human umbilical vein endothelial cells which over-express VCAM-1 were cultured. To detect the microbubbles＇ targeting function, the specific binding of immunomicrobubbles to the activated endothelial cells was observed in vitro. Results The prepared immunomicrobubbles were positive in slide agglutination test. The activated endothelial cells were surrounded by plenty of anti-VCAM-1 conjugated microbubbles. While there was limited binding of control bubbles to normal or activated endothelial cells. Conclusions The antibody can be effectively attached to the surface of albumin microbubbles by SPDP conjugation technology. And the studies in vitro had proved that the prepared immunomicrobubble could specifically bind to the targeted cells.