The Specific Isolation Of Immunoglobulin From Human Serum By Using Magnetic Polymer Beads Surface Modified With 2-mercaprydime

Immunoglobulins have been widely applied in biotechnology, biomedicine and clinical therapy as the most kind of antibodies in human serum. Although a large number of methodologies have been researched, involving precipitation methods, electrophoretic separations, membrane filtration and liquid chromatography, their complicated equipment, costly materails and inconvenient pretreatment of the samples confine these methods ideal only for small-scale separations. And in recently, the magnetic polymer beads became a high efficient protocol in protein purification for its high abundant surface area and sensitive magnetism responsiveness.So, thiophilic functional groups were grafted in these magnetic polymer beads to purify immunoglobulin from human serum directly.It is a novel method to isolate immunoglobulin with high efficiency, high purity and strong biological activity. The results are listed in below.1. The paramagnetic Fe3O4 magnetic particles were prepared by the oxidation and reduction action and their surface was modified by Oleic Acid. Then, these hydrophobic magnetic particles were dispersed in cyclohexane to be a whole magnetic fluid.2. Two kinds of magntic polymer beads were respectively synthesized by miniemulsion polymerization with DVB/VAc and St/DVB/VAc. After complete alcoholysis, thiophilic function group(2-mercaprydime) was introduced on the surface of these magnetic beads by Michael addition reaction. The morphology, structure and composition of these magnetic polymer beads were analyzed by using FTIR, SEM, element analysis. It indicated the magnetic beads prepared with St/DVB/VAc was more suitable to isolate immunoglobulin from human serum.3. The process of the isolation to purify immunoglobulin from human serum by magnetic beads was detected by using SDS-PAGE, Native-PAGE, and HPSEC. The result shows that the isolated immunoglobulin has a high purity.The relative factors to influence the isolation of immunoglobulin by using magnetic microbeads, such as ionic species and concentration in absorbed buffer and elution, the responding pH in these buffers, the desired time for adsorption and desorption and the suitab. ration between the thiophilic magnetic polymer beads and human serum, the responding temperature for the adsorption and the desorption, were all detailedly studied. The optimal processes to isolate immunoglobulin were achieved. The adsorption should be carried out in low salt concentration and at near neutrality. The desorption could be performed in high chloride ion concentration and high pH at 30℃.