MR对比剂荧光VEGF165-USPIO合成及其活性的体外实验

The synthesis of CY5.5-VEGF165-USPIO and its activity——an in vitro study

目的:探讨荧光VEGF165-USPIO(血管内皮生长因子165-超顺磁性氧化铁)作为MR对比剂的可行性,检测该对比剂体外对VEGF165抗原的靶向性。方法:通过氨基-羧基偶联方式将VEGF165单克隆抗体和USPIO连接,然后将Cy5.5标记到单克隆抗体表面,构建Cy5.5-anti-VEGF165-USPIO靶向对比剂。以离心和磁分离器的洗涤手段进行改良ELISA实验检测其体外活性,实验组加入0.25ml的荧光-anti-VEGF165-USPIO,对照组加入相同量的荧光USPIO,每组各设5个离心管,两样本为计量资料,采用独立t检验比较不同组间吸光值差异有无显著性。结果:通过分光光度计验证对比剂连接成功,VEGF165单克隆抗体偶联率达到了71.7%,CY5.5的偶联率为83.3%。ELISA实验表明实验组和对照组各自吸光值差异有显著性意义(P<0.05),实验组OD(吸光值)明显高于对照组。结论:荧光anti-VEGF165-USPIO体外有结合VEGF165的活性,为进一步的MR肿瘤靶向对比剂研究奠定了实验基础。

Objective：To explore the feasibility of the synthesis of a MR contrast agent Fluorescence VEGF165-US-PIO and to establish an ELISA method for detecting the targeting of this agent to VEGF165-USPIO antigen in vitro. Methods：By applying the coupling of amino-carboxyl,the VEGF165 monoclonal antibody and USPIO were coupled.Then the Cy5.5 was marked to the surfac of monoclonal antibody,and Cy5.5 anti-VEGF165-USPIO targeting contrast agent was established.The centrifuge and magnetic separation were used as the washing solution.Two groups were set up.In the test group,fluorescence anti-VEGF165-USPIO （0.25mL）was used,while in the control group the same amount of fluorescence-USPIO was applied.Each group had five centrifugal pipes.By applying the two-sample independent T-test,we compared the data obtained from both washings.Results：The new type of magnetic resonance contrast agent was successfully combined. Coupling efficiency of VEGF165 monoclonal antibody was 71.7%,and CY5.5 was 83.3%.ELISA assay showed that ab-sorbance values of the test group and the control group were different （P 〈0.05 ）,while the test group （absorbance） showing a significantly higher OD （absorbance ）.Conclusion：Fluorescent anti-VEGF165-USPIO has activity to bind VEGF165 in vitro.