表面改性的纳米Fe3O4颗粒用于抗血清快速分离纯化

Rapid Separation of Antiserum by Surface Modified Fe3O4 Nano-particles

摘要:

采用硅烷化试剂Si(OC2H5)3C3H6NH2(APTES)对纳米Fe3O4颗粒表面进行氨基化改性后, 考察了不同浓度偶联剂戊二醛对于颗粒表面固定牛血清白蛋白(BSA)量的影响。此超顺磁性免疫铁颗粒(SPIO)加入兔抗BSA血清中特异性结合BSA抗体后, 用Gly-HCl缓冲液洗脱得到IgG。结果表明当戊二醛浓度大于10%时, 单位颗粒固定蛋白的量达到最大值约140 mg/mg, 10 min, 15 mg的SPIO即可将1 mL抗血清完全分离, 经过两次快速洗脱, 颗粒表面吸附的抗体即可得到纯化; 琼脂扩散实验表明分离后的抗体仍保持较高活性, SDS-PAGE电泳结果表明用此方法纯化后的兔抗BSA IgG纯度大于99%, 比传统的(NH4)2SO4法有了较大提高, 但纯化量并没有减少; SPIO在经过五次重复利用后仍能保持78%以上的分离效果。

Abstract:

The nano-particles’ surface were modified by silane reagent Si(OC2H5)3C3H6NH2. Bovine serum albumin (BSA) was coupled by a series of glutaraldehyde concentration. Under optimal reaction conditions the maximum amount of immobilized BSA was 140 mg/mg Fe3O4, when glutaraldehyde concentration was above 10%. After immersing in anti-BSA rabbit serum and regenerating by Gly-HCl buffer, the purity of IgG by super-paramagnetic iron oxide (SIPO) nano-particles was characterized by UV-spectrometer, SDS-PAGE and immuno-diffuse. Results showed that subtotal separation was achieved at 10 min, 15mg SPIO in 1ml serum. The purity was much higher than that with the traditional (NH4)2SO4 method. The activity of special protein probe maintained above 78% after 5 consecutive uses.