新型免疫亲和嗜硫整体色谱材料的制备与性能研究

At present, biological affinity ligands were used to realize the antibody concentration and purification in most condition. Polymeric monolith became a substitute for conventional biological affinity ligands, and it not only cans lower the cost but also make the storage condition became milder. According to the above problem, this project is to prepare a series of novel immuno-affinity monolithic materials for the purification of antibody by the method of one-pot or post-modification. The antibodies in the complex sample will be enriched and purified by the specific affinity interaction between the group of ether-sulfone and antibody. The synthesis route would be tested, the method for the monolith preparation wolud be investigated. Especially, the stability of the monolith would be improved. As an abiotic affinity ligands, the thiophilic chromatographic material can stored in room-temperature environment, and has an unparalleled advantages in stability and regeneration. In addition, monolithic thiophilic chromatographic material can be coupled with micro-column high performance liquid chromatography, and the condition of enrichment and purification for antibody will be optimized, loading and exclusion buffer will be selected, the purification efficiency will be increased and its mechanism will be explored. The project will provide low-cost and easy-operation new material and high-efficiency technology for the separation, enrichment and purification of antibody.

目前，抗体的分离、富集与纯化方式大多使用基于生物亲和配基的亲和色谱法。而多数的生物亲和配基存在制备成本高、存储条件苛刻等不足之处。本项目拟以嗜硫试剂为功能单体，采用原位聚合、后修饰等方法制备一系列价格低廉、稳定性高、再生性好的新型免疫亲和嗜硫整体色谱材料。深入考察不同的制备方法对整体材料稳定性能的影响；探讨材料中的嗜硫基团与抗体间特异性亲和吸附的原理与规律；优化其与微柱液相色谱等自动化仪器联用时上样与洗脱的条件，提高抗体纯化效率，最终实现复杂体（如血清、血浆）中抗体的分离、富集与纯化。本项目的成功实施将为抗体的分离、富集与纯化提供新材料和新技术。

本项目以乙烯基砜（DVS）、磺胺(SAA)、璜酰胺(SFA)等嗜硫试剂为功能单体，采用原位聚合、后修饰等方法，利用双键聚合、环氧开环等原理制备了一系列价格低廉、稳定性高、再生性好的新型免疫亲和嗜硫色谱整体柱，包括：有机高分子聚合免疫亲和整体柱：SAA-poly(GMA-co-MBAA)、poly(TEPIC-co-MBAA)、poly（DVS-co-MBAA）、poly(MBAA-co-DVS-MPCA)，有机-无机杂化新型免疫亲和嗜硫整体柱poly（DVS-co-TMTVS）；嗜硫色谱硅球SiO2@p(TEPIC-co-SAA)；嗜硫磁珠Fe3O4@SiO2@SFA。深入考察了不同的制备方法对嗜硫材料稳定性能的影响；探讨了材料中的嗜硫基团与含二硫键化合物抗体间特异性亲和吸附的原理与规律；优化了材料与微柱液相色谱联用时上样与洗脱的条件，提高了抗体纯化效率，并最终实现在有干扰物质胰岛素存在的条件下，对抗体的分离、富集与纯化。本项目的成功实施将为抗体的分离、富集与纯化提供新材料和新技术。