Construction and measuring combination of KDR-targeted ultrasound contrast agent in vitro for evaluating endometrial receptivity

摘要

Objective: To investigate the preparation of a new kind of targeted lipid ultrasound contrast agent with anti-KDR antibody based on biotin-avidin bridge (MB-BAB-KDR) which could combine specifically with KDR that increases during the time of embryo implantation. Then its binding capability in vitro was evaluated. Materials and Methods: The agitation of high-speed method was employed to prepare biotin-microbubbles (MB-B), and biotin avidin mediated technique was used to produce MB-BAB-KDR. MB-BAB-KDR, MB-B, and biotin-microbubbles-KDR (MB-B-KDR) were incubated with fluorescein-conjugated affiniPure goat anti-rat IgG (H+L) to assess the linked condition. Second, MB-BAB-KDR and control groups (MB-B and MB-B-KDR) were incubated with human umbilical vein endothelial cell (HUVEC). Rosette formation rate was observed and calculated. Then, the parallel plate flow chamber technology was used to access attachment efficiency to KDR Fc. Results: The surface of bubbles could carry KDR antibody through "biotin-avidin" bridge. After incubated with second antibody, bright green fluorescence (II grade) could be observed in MB-BAB-KDR group, as compared with weak fluorescence in control groups of MB-B (0 grade) and MB-B-KDR (I grade). The surrounding rosette formation rate on HUVEC was 89.86% in MB-BAB-KDR group and that of control groups were 7.13% (MB-B-KDR) and 3.02% (MB-B) (p < 0.05). The number of MB-BAB-KDR bound to KDR Fc increased as the KDR Fc density increased (p < 0.05). Under the same concentration, the MB-BAB-KDR bound to KDR Fc increased as time extended. Conclusion: The successful preparation of MB-BAB-KDR with anti-KDR antibody which shows specially targeting binding capability with HUVEC and stability in shear stress may be served as a noninvasive detection of endometrial vascular KDR expression and provide an experimental foundation for evaluating endometrial receptivity in vivo.