Specific Isolation Of Immunoglobulin G Directly From Human Serum By Using Thiophilic Magnetic Polymer Microspheres

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.