嗜硫性磁性微球特异性分离人血清免疫球蛋白G
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摘要
随着抗体免疫技术广泛应用在生物化学、生物医药等生产领域,人们对免疫抗体的提纯效率、纯度、成本、活性等方面也提出了更高的要求。由于传统的纯化方法分离效率低、操作复杂、分离周期较长、生产成本高、生物活性损失较大,因此,开发一种快速、高效、简易、低成本制备高纯度免疫球蛋白的方法和工艺成为各国学者关注的热点。本文采用细乳液聚合技术制备了PS-Fe3O4磁性复合微球,通过偶联恰当的特征配合基,实现了对免疫球蛋白G的特异性识别,利用磁分离技术,直接从人血清中分离、纯化人体免疫球蛋白G。在分离过程中,主要讨论了血清与磁性微球的比例、吸附溶液和解吸附溶液的pH值、解吸附溶液的总体积以及离子强度变化、吸附和解吸附的时间变化等因素对分离的影响,并通过高效液相排阻色谱法(HPSEC)和BCA试剂盒测定蛋白法等分别进行了定性和定量分析,证明了此方法的可行性与可靠性,为分离纯化IgG提供了一种新的方法。主要研究结果如下:
1.用化学共沉淀法制备了超顺磁性、表面亲油性的Fe3O4磁流体,其平均粒径小于17.7nm;通过细乳液聚合法实现苯乙烯单体对磁粒子的有效包裹,制备了平均粒径为120nm、分散均匀的磁性聚合物微球,其磁含量大于20%,饱和磁强度达到32.3emu/g。通过高分辨透射电镜证实了合成的磁性聚合微球为核壳式结构。
2.在合成磁性微球过程中,引入酯官能团(-OOC2H5),因此在磁球表面可以修饰一定浓度的配体(2-巯基烟酸),实现磁性微球对免疫球蛋白G(IgG)的特异性识别与分离,分离效果显著。
3.研究了磁球分离血清的各种影响因素,结果表明当配基的浓度为0.232mmol/g时,其适宜的范围是:血清与磁性微球的适合比例为1.0~1.67mL/g、吸附溶液的pH值为6.0~7.5、解吸液的pH值为12~13.5、吸附以及解吸附所需时间仅为5min、解吸溶液的体积为15~25mL、解吸溶液可适当添加0.5mol/L的Cl—溶液来促进解吸附。
4.在磁性微球对血清中的IgG进行批量分离中,所提纯的IgG生物活性高、纯度较高,而且在整个分离工艺中,磁性微球技术表现出分离效率高、分离周期短、分离条件温和、操作简便等优点,为高效、高纯度、高活性分离提纯免疫球蛋白G提供了一种新的方法,在大规模、工业化生产方面具有一定的潜力。
The growing interests and needs for the antibodies at the forefront of biotechnology and biomedicine are greatly prompting the development of the more effective and efficient technologies to isolate the immunoglobulin with higher purity, higher biological activity and lower cost.
Although the immunoglobulin G has been isolated from human serum by many technologies, the low efficiency of separation, complicated operation, long period of purification, high cost of production, low purity and weak biological activity become more and more limitative for its extensive application in biotechnology. Thus, considerable interests are being focused on the development of a new method to isolate IgG with more efficient and more selective.
In this thesis, the responding magnetic beads of Fe3O4/PS were prepared by miniemulsion polymerization and utilized to isolate IgG directly from human serum.
In the process of the isolation, the main factors to influence the isolated efficiency, such as the suitable proportion between human serum and magnetic microspheres, the pH values of adsorption and desorption solution, the ion intensity of the desorption solution, the selective absorption period and the responding desorption period, were all discussed. Moreover, both quantitative and qualitative analyses were carried by high performance size-exclusion chromatography (HPSEC) and BCA protein assay kit. It indicated that the method to isolate IgG directly from the human serum by using magnetic microspheres was much effective and rather feasible.
Major research results as following:
1. The paramagnetic and hydrophobic Fe3O4 magnetic fluid was prepared by the oxidation and reduction action between the ferric chloride and ferrous chloride. The average diameter of these magnetite particles was less than 17.7nm. The uniformly superparamagnetic polymer beads with high magnetic content were prepared with styrene, divinyl benzene and vinyl acetate by miniemulsion polymerization. The average particle diameter was smaller than 120nm and the magnetic content was more than 20%. The saturated magnetization reaches 32.3emu/g. The evident core-shell structure was also observed by using high-resolution TEM.
2. The functional groups of ester were introduced to the surface of the magnetic microspheres by the copolymerization of styrene and vinyl acetate. Then, the divinyl sulphone was added to activate the particles and 2-mercaptonicotinic acid was grafted on the magnetic beads to endow these particles with the ability to selectively recognize the human IgG. These magnetic beads were successfully applied to isolate the antibodies directly from human serum by the assistance of the external magnetic field.
3. Main factors to influence the isolation of IgG by using magnetic microbeads were discussed in detail. Here are the results: when the surface concentration of thiophilic ligands arrived 0.232mmol/g, appropriate range of the ratio (magnetic microspheres and serum) was found from 1.0mL/g to 1.67mL/g; The process of the isolation was heavily influenced by the pH values of the buffer solution, and it was found that the suitable pH range of the adsorbed solution should fluctuate from 6.0 to 7.5 and the responding pH range for the desorption solution varied from 12 to 13.5; Five minutes was enough for the magnetic mircrospheres to selectively absorb IgG from the human serum, and the period of the desorption of IgG from the magnetic microspheres was also very short.
4. During the batch separation of IgG directly from the human serum by magnetic microspheres, not only the purity of the isolated IgG was greatly improved but also the biological activity of IgG was completely kept due to the mild conditions in the process of the isolation. The prominent advantages of this technology was observed that the high separation efficiency, short period (less than 2h), mild condition (separated at 0℃, pH value of its absorption environment is 7), simple operation (can be repeated easily and conveniently), conventional equipments and so on. This just provided another approach to isolate IgG from human serum with high efficiency, high purity and strong bioactivity. Therefore, this technology possesses some potentiality in industrial area for mass production.
1.用化学共沉淀法制备了超顺磁性、表面亲油性的Fe3O4磁流体,其平均粒径小于17.7nm;通过细乳液聚合法实现苯乙烯单体对磁粒子的有效包裹,制备了平均粒径为120nm、分散均匀的磁性聚合物微球,其磁含量大于20%,饱和磁强度达到32.3emu/g。通过高分辨透射电镜证实了合成的磁性聚合微球为核壳式结构。
2.在合成磁性微球过程中,引入酯官能团(-OOC2H5),因此在磁球表面可以修饰一定浓度的配体(2-巯基烟酸),实现磁性微球对免疫球蛋白G(IgG)的特异性识别与分离,分离效果显著。
3.研究了磁球分离血清的各种影响因素,结果表明当配基的浓度为0.232mmol/g时,其适宜的范围是:血清与磁性微球的适合比例为1.0~1.67mL/g、吸附溶液的pH值为6.0~7.5、解吸液的pH值为12~13.5、吸附以及解吸附所需时间仅为5min、解吸溶液的体积为15~25mL、解吸溶液可适当添加0.5mol/L的Cl—溶液来促进解吸附。
4.在磁性微球对血清中的IgG进行批量分离中,所提纯的IgG生物活性高、纯度较高,而且在整个分离工艺中,磁性微球技术表现出分离效率高、分离周期短、分离条件温和、操作简便等优点,为高效、高纯度、高活性分离提纯免疫球蛋白G提供了一种新的方法,在大规模、工业化生产方面具有一定的潜力。
The growing interests and needs for the antibodies at the forefront of biotechnology and biomedicine are greatly prompting the development of the more effective and efficient technologies to isolate the immunoglobulin with higher purity, higher biological activity and lower cost.
Although the immunoglobulin G has been isolated from human serum by many technologies, the low efficiency of separation, complicated operation, long period of purification, high cost of production, low purity and weak biological activity become more and more limitative for its extensive application in biotechnology. Thus, considerable interests are being focused on the development of a new method to isolate IgG with more efficient and more selective.
In this thesis, the responding magnetic beads of Fe3O4/PS were prepared by miniemulsion polymerization and utilized to isolate IgG directly from human serum.
In the process of the isolation, the main factors to influence the isolated efficiency, such as the suitable proportion between human serum and magnetic microspheres, the pH values of adsorption and desorption solution, the ion intensity of the desorption solution, the selective absorption period and the responding desorption period, were all discussed. Moreover, both quantitative and qualitative analyses were carried by high performance size-exclusion chromatography (HPSEC) and BCA protein assay kit. It indicated that the method to isolate IgG directly from the human serum by using magnetic microspheres was much effective and rather feasible.
Major research results as following:
1. The paramagnetic and hydrophobic Fe3O4 magnetic fluid was prepared by the oxidation and reduction action between the ferric chloride and ferrous chloride. The average diameter of these magnetite particles was less than 17.7nm. The uniformly superparamagnetic polymer beads with high magnetic content were prepared with styrene, divinyl benzene and vinyl acetate by miniemulsion polymerization. The average particle diameter was smaller than 120nm and the magnetic content was more than 20%. The saturated magnetization reaches 32.3emu/g. The evident core-shell structure was also observed by using high-resolution TEM.
2. The functional groups of ester were introduced to the surface of the magnetic microspheres by the copolymerization of styrene and vinyl acetate. Then, the divinyl sulphone was added to activate the particles and 2-mercaptonicotinic acid was grafted on the magnetic beads to endow these particles with the ability to selectively recognize the human IgG. These magnetic beads were successfully applied to isolate the antibodies directly from human serum by the assistance of the external magnetic field.
3. Main factors to influence the isolation of IgG by using magnetic microbeads were discussed in detail. Here are the results: when the surface concentration of thiophilic ligands arrived 0.232mmol/g, appropriate range of the ratio (magnetic microspheres and serum) was found from 1.0mL/g to 1.67mL/g; The process of the isolation was heavily influenced by the pH values of the buffer solution, and it was found that the suitable pH range of the adsorbed solution should fluctuate from 6.0 to 7.5 and the responding pH range for the desorption solution varied from 12 to 13.5; Five minutes was enough for the magnetic mircrospheres to selectively absorb IgG from the human serum, and the period of the desorption of IgG from the magnetic microspheres was also very short.
4. During the batch separation of IgG directly from the human serum by magnetic microspheres, not only the purity of the isolated IgG was greatly improved but also the biological activity of IgG was completely kept due to the mild conditions in the process of the isolation. The prominent advantages of this technology was observed that the high separation efficiency, short period (less than 2h), mild condition (separated at 0℃, pH value of its absorption environment is 7), simple operation (can be repeated easily and conveniently), conventional equipments and so on. This just provided another approach to isolate IgG from human serum with high efficiency, high purity and strong bioactivity. Therefore, this technology possesses some potentiality in industrial area for mass production.
引文
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