基于EDC/sulfo-NHS的羧基化SPIO表面抗体耦联
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江苏省生物材料与器件重点实验室开放基金(2012LBMD05);环境医学工程教育部重点实验室开放基金(2012EME005)


Conjugation of antibody on carboxyl-SPIO nanoparticles based on EDC/sulfo-NHS
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    目的:研究羧基化超顺磁性氧化铁纳米颗粒(SPIO)纳米粒子与anti-EMMPRIN单克隆抗体耦联的有效方法,考察不同pH条件下进行耦联反应的效率及anti-EMMPRIN-SPIO的粒径水平。方法:用EDC/sulfo-NHS法将anti-EMMPRIN单克隆抗体耦联到羧基化SPIO纳米粒子表面,用动态光散射法检测所得anti-EMMPRIN-SPIO的粒径,并用Bradford法和SDS-PAGE法分析不同酰胺化pH条件下的耦联效率。结果:通过EDC/sulfo-NHS的作用,anti-EMMPRIN单克隆抗体有效地耦联到羧基化SPIO表面。pH7.8时酰胺化的anti-EMMPRIN-SPIO纳米粒子的粒径最小,为63.15 nm。pH6.8和pH7.3条件下酰胺化所得上清中的抗体含量较低,分别为1.943 2 × 10-4 g/L和3.511 1 × 10-4 g/L,SDS-PAGE凝胶图谱上可明显观察到pH6.8-pH7.3和pH7.8条件下所制备样品的谱带分别位于55 000 和25 000。结论:EDC/sulfo-NHS法是anti-EMMPRIN单克隆抗体耦联于羧基化SPIO表面的有效方法。在pH6.8-pH7.3-pH7.8条件下酰胺化耦联效率较高,所制备的anti-EMMPRIN-SPIO粒径较小。pH值对EDC/sulfo-NHS介导的耦联反应的耦联效率及anti-EMMPRIN-SPIO的粒径有较大影响。

    Abstract:

    Objective:To conjugate anti-EMMPRIN monoclonal antibody on the surface of carboxyl-SPIO nanoparticles. The efficiency of superparamagnetic iron oxide (SPIO) conjugated with anti-EMMPRIN monoclonal antibody and the size of anti-EMMPRIN-SPIO were investigated in different pH values. Methods:Anti-EMMPRIN monoclonal antibody conjugated SPIO nanoparticles were performed through a coupling strategy of EDC and sulfo-NHS. Bradford assay and SDS-PAGE analysis were used for measuring the conjugation efficiency. The size of anti-EMMPRIN-SPIO was evaluated by dynamic light scattering method. Results:The smallest diameter of 63.15 nm was obtained when pH 7.8 was used. The lower concentration of antibody in supernatant with pH6.8 and pH7.3 was 1.943 2 × 10-4 g/L and 3.511 1 × 10-4 g/L, respectively. Anti-EMMPRIN mAb was detected in lanes of pH6.8, pH7.3 and pH7.8 which revealed two bands with weight of 55 000 and 25 000. Conclusion:EDC/sulfo-NHS method is an efficient method for conjugating anti-EMMPRIN monoclonal antibody on SPIO nanoparticles. Under the amidation condition with pH value of 6.8, 7.3 and 7.8, higher conjugation efficiency and smaller size of anti-EMMPRIN-SPIO were obtained. The results showed that pH value is a very important factor which influences conjugation efficiency and the size of anti-EMMPRIN-SPIO.

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刘 琳,张小强,张 宇,浦跃朴,尹立红,刘 卉,徐 靓,杨文倩,徐春雨.基于EDC/sulfo-NHS的羧基化SPIO表面抗体耦联[J].南京医科大学学报(自然科学版),2013,(4):480-484

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  • 收稿日期:2012-10-19
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  • 在线发布日期: 2013-04-29
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