壳聚糖磁性复合微球的制备、表征及其在T细胞富集中的应用

发布时间：2018-06-20 07:22

本文选题：免疫磁性微球 + 壳聚糖　；参考：《暨南大学》2005年硕士论文

【摘要】：免疫磁性微球(Immunomagnetic microspheres简称IMM)是将单抗或者生物活性物质通过共价交联或物理吸附包被到有磁性高分子复合微球的表面,形成具有免疫活性的磁性微球。免疫磁性分离是基于细胞表面抗原以及某种蛋白与连有磁性微球的特异性单抗相结合,在外加磁场的条件下,通过抗体与IMM相连的细胞和目标蛋白被吸附而滞留在磁场中,由于微球表面的单抗只结合含有特异性抗原的细胞和蛋白,从而达到与其他种类的细胞和蛋白相分离的目的。免疫磁性分离的方法与常用的细胞分离方法(FACS法、二抗致敏红细胞法、与组织培养管吸附的抗体结合法等)相比,有方便、快速、低毒、高效的优点,而迅速渗透到免疫、生理、病理、药理、微生物等领域。本文从壳聚糖磁性复合微球的制备出发,在以下几个方面取得一定的进展: 1.用反相微乳的方法合成了CMCM(Chitosan magnetic composite microspheres简称CMCM),对粒径和表观形貌、微球铁含量、磁响应性、表面官能基团等性质进行了表征,与单体聚合法制备的聚苯乙烯磁性复合微球(Polystyrene magnetic composite microspheres,简称PMCM)进行比较后得知,CMCM是比PMCM更理想的免疫磁性微球载体材料,有可能成为一种蛋白吸附性能良好的磁性高分子复合微球。 2.探讨了不同条件下CMCM的蛋白吸附条件,在此基础上初步探讨了免疫磁性微球的制备和细胞分离富集的可行性。活化后的CMCM对小牛血清蛋白(BSA)的吸附性能较活化前提高了近5倍(从5.4mg/g到24.6mg/g)。时间对吸附量影响甚微,温度对吸附行为影响也很小,离子浓度对活化的磁性微球的吸附有一定的影响,随着NaCl浓度逐渐增加至1.0mol/l,微球对BSA的吸附逐渐降低,因此应尽量避免在离子浓度较高的条件下进行吸附操作。 3.把制备的CMCM经过戊二醛的活化,再连接鼠抗人CD_3单抗制备了CD_3免疫磁性微球,用所制备鼠抗人CD_3单抗的免疫磁性微球可以特异性地从人全血中分离出CD_3~+细胞,分离纯度经流式细胞仪检测均达到95%以上。证明了CMCM作为免疫磁性微球载体的可行性。
[Abstract]:Immunomagnetic microspheres (imm) is a kind of immunoreactive magnetic microspheres, which is coated on the surface of magnetic polymer composite microspheres by covalent crosslinking or physical adsorption of monoclonal or bioactive substances. Immunomagnetic separation is based on the binding of a cell surface antigen and a protein to a specific monoclonal antibody attached to a magnetic microsphere. Under the condition of an external magnetic field, cells and target proteins linked to imm by antibodies are adsorbed in the magnetic field. Because the McAbs on the surface of the microspheres only bind to the cells and proteins containing specific antigens, they can be separated from other kinds of cells and proteins. The method of immunomagnetic separation is more convenient, faster, less toxic and more efficient than the usual methods of cell separation, second antibody sensitized red blood cell, antibody binding to tissue culture tube, and so on. Physiology, pathology, pharmacology, microbiology and other fields. Based on the preparation of chitosan magnetic composite microspheres, some progress has been made in the following aspects: 1. CMCM-chitosan magnetic composite microspheres was synthesized by inverse microemulsion method. The particle size and morphology, iron content, magnetic response and surface functional groups were characterized. Compared with the polystyrene magnetic composite microspheres, prepared by monomer polymerization, the results show that the polystyrene magnetic composite microspheres are more ideal than PMCMs in immunomagnetic microspheres. It is possible to become a kind of magnetic polymer composite microspheres with good protein adsorption performance. 2. The protein adsorption conditions of CMCM under different conditions were discussed, and the feasibility of preparation and cell separation and enrichment of immunomagnetic microspheres was preliminarily discussed. The adsorption performance of activated CMCM for bovine serum protein (BSA) was nearly five times higher than that before activation (from 5.4mg/g to 24.6 mg / g). Time had little effect on the adsorption amount and temperature had little effect on the adsorption behavior. Ion concentration had a certain effect on the adsorption of activated magnetic microspheres. With the increase of NaCl concentration to 1.0 mol / l, the adsorption of BSA on the microspheres gradually decreased. Therefore, the adsorption operation should be avoided under the condition of high ion concentration as far as possible. 3. The prepared CMCM was activated by glutaraldehyde and then connected with mouse anti-human CDS3 monoclonal antibody to prepare CDS3 immunomagnetic microspheres. CD3 cells could be specifically isolated from human blood by using immunomagnetic microspheres prepared by mouse anti human CD3 McAb. The purity was more than 95% by flow cytometry. The feasibility of CMCM as a carrier of immunomagnetic microspheres was proved.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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