基于EDC/sulfo-NHS的羧基化SPIO表面抗体耦联

Conjugation of antibody on carboxyl-SPIO nanoparticles based on EDC/sulfo-NHS

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 2×10-4g/L和3.511 1×10-4g/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。

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^-4g/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.