用于单克隆抗体纯化的仿生多肽超大孔PGMA微球制备及其性能

以仿生多肽配基FYEILHC为亲和配基、以葡聚糖修饰的聚甲基丙烯酸缩水甘油酯[Dextran-poly(glycidyl methacrylate), Dextran-PGMA]超大孔微球为基质,制备用于单克隆抗体纯化的仿生多肽超大孔PGMA微球,在环氧氯丙烷中滴加2 mol/LNaOH使其表面衍生出环氧基,在表面修饰FYEILHC;用扫描电镜表征微球表面形貌,用AKTA蛋白纯化系统考察了Dextran-PGMA微球和琼脂糖微球对抗体的动态吸附量随线性流速的变化。结果表明,偶联FYEILHC后Dextran-PGMA微球仍能保持其大孔结构,在923 cm/h线性流速下,其对抗体的动态吸附量仅下降约8%,而琼脂糖微球的动态吸附量则迅速下降25%。表明在较高流速下,抗体在Dextran-PGMA微球上的传质性能较好。吸附?用0.1 mol/L NaOH原位清洗重复40次后,Dextran-PGMA微球对抗体的动态吸附量约为(21?1) mg/mL,表明微球具有良好的化学稳定性;血清中回收的抗体纯度为95.0%,表明仿生多肽亲和介质具有从复杂生物样品中纯化抗体的巨大潜力,可满足高流速、高通量抗体分离纯化需求。     

磁性高分子微球的制备及其应用

介绍了有机高分子与磁性氧化铁复合的制备方法,总结了磁性高分子微球在废水处理、生物工程方面的应用,并对其发展趋势做了展望。     

抗体药物分离纯化中的层析技术及进展

抗体,特别是单克隆抗体是一类重要的生物技术药物,具有巨大的市场前景。目前抗体药物生产的瓶颈已由抗体表达转移到抗体的分离纯化中,其中层析是最关键的技术。典型的抗体纯化过程首先通过蛋白A亲和层析对抗体进行捕获,然后进一步精制,主要方法包括离子交换层析、疏水相互作用层析以及羟基磷灰石层析等,这些传统的层析方法不可避免存在一定的局限性。为此,研究者致力于开发合适的新方法,如疏水性电荷诱导层析和短肽仿生层析等,很好地弥补了传统层析方法的不足。本文根据近年来国内外在单抗药物分离纯化方面所取得的研究进展,着重介绍了在单抗分离纯化中的常用的层析技术及研究进展。     

亲和仿生层析及在抗体纯化中的应用

亲和仿生层析是一种新型的生物分离技术,可用于生物活性物质分离,尤其在抗体纯化中表现出良好的应用前景,具有价格低廉、结构稳定、特异性较高等优点。亲和仿生层析的核心是具有特定结构的仿生配基,主要包括化学合成配基和短肽配基两种,通常通过合理的手段进行筛选和设计。理性设计是一种行之有效的方法,针对一个特定的目标蛋白,随机地去选择配基是不可能的,必须采用理性设计构建一个合理的配基库,并通过高通量的筛选技术,才能得到合适的仿生配基。本文根据近年来国内外亲和仿生层析的研究进展,着重介绍了亲和仿生配基的筛选和设计以及在抗体纯化中的应用。     

羧基磁性微球的制备及其用于CD4细胞分离应用

采用溶液聚合法制备具有良好悬浮性和磁响应性的羧基聚丙烯酰胺磁珠,对羧基磁性微球的形貌、结构、悬浮稳定性进行表征.研究结果显示,羧基聚丙烯酰胺磁性微球的平均粒径为20nm,粒径分布比较均匀,近似为球形的壳核结构,核为磁性基质,壳为羧基聚丙烯酰胺;将羧基聚丙烯酰胺磁珠用于CD4细胞分离研究表明,分选后的细胞纯度高达94％.     

Preparation and characterization of superparamagnetic chitosan microspheres: Application as a support for the immobilization of tyrosinase

Superparamagnetic chitosan microspheres were prepared by a water‐in‐oil suspension‐crosslinking technique. To this end, magnetite particles were dispersed in a chitosan solution in acetic acid. The dispersion was added to toluene containing Span 20 as a surfactant with stirring. Chitosan solution droplets were hardened with glutaraldehyde. The magnetic chitosan microspheres obtained were characterized with scanning electron microscopy, differential thermal analysis, and vibrational magnetometry. The microspheres had a wide size distribution, ranging from 43 ± 25 to 255 ± 55 μm, that depended on the reaction conditions. The mean particle size decreased with an increase in the concentration of Span 20 or the amount of glutaraldehyde and with the addition of NaCl. However, a major size reduction was achieved by an increase in the stirring rate. Tyrosinase was immobilized on the microspheres. The immobilized enzyme retained 70% of its activity, as determined by the capacity to degrade phenolic compounds. The immobilized tyrosinase resulted in greater stability than the free enzyme. In addition, the enzyme maintained 65% of its phenol oxidation activity after 10 cycles of reuse. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 651–657, 2005     

Preparation of highly magnetic chitosan particles and their use for affinity purification of enzymes

A novel protocol for preparation of highly magnetic chitosan particles, with a coercive force as high as 3500 Oe, has been developed. The surface of the particles was functionalized with aldehyde groups to facilitate the attachment of affinity ligands. The optimum conditions for the preparation of highly magnetic chitosan particles and immobilization of trypsin on magnetic particles were obtained; these particles were then used for affinity purification of aprotinin, and the conditions of affinity purification are discussed in detail. The proposed method was successfully applied to the affinity purification of aprotinin. Copyright © 2003 Society of Chemical Industry     

Magnetic polymer microspheres with azidocarbonyl groups: Synthesis,characterization and application in protein immobilization

A novel magnetic polymer microsphere with amide groups and carboxyl groups was synthesized and reported here. The azidocarbonyl groups were derived from amide groups and linked to the proteins to investigate their immobilization capacity. The morphology, size, functional groups and magnetic properties of magnetic microspheres were characterized by optical microscopy, particle size analyzer, atom force microscopy, magnetic force microscopy, fourier transform infrared spectrometer, vibrating‐sample magnetometer and thermal gravimetric analysis. The results indicated that the magnetic polymer microspheres had a well spherical shape with the size ranging from 1 to 10 μm, highly reactive functional groups, superparamagnetism and strong magnetic responsibility with saturation magnetization of 18.443 emu/g and Fe₃O₄ content around 21%. The immobilization capacity (η) was over 70%. The novel azidocarbonyl magnetic polymer microspheres showed potentials to be a good magnetic support and promising applications in bioseparation and biomedical fields. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

混合模式介质的制备及用于清除抗体中蛋白A

针对抗体纯化过程中脱落蛋白A的清除,设计和制备了以N-苄基-N-甲基乙醇胺为功能配基的混合模式层析介质Adhere NUPharose FF,该配基兼有疏水、静电和氢键作用。采用烯丙基缩水甘油醚活化法,并对琼脂糖微球基质的活化和配基偶联条件进行了优化,优化后活化密度达260 μmol·ml^-1以上,配基密度达120 μmol·ml^-1。同时以抗体和蛋白A的混合物为研究对象来考察该介质对抗体中蛋白A的清除能力,结果表明对蛋白A的有效去除率达到83.1%,为抗体制品中蛋白A的清除提供了一种可行性方法。     

丹皮酚磺酸钠的合成及精制

目的：提高丹皮酚磺酸钠制备的收率,并提供一种新的精制方法。方法：以氯磺酸做磺化试剂,乙酸乙酯做溶剂,60~C／~应1h,与氯化钠成盐后,经活性炭脱色,乙醇精制,得到高纯度的丹皮酚磺酸钠。结果：合成了有关物质、氯离子、硫酸根离子均合格的丹皮酚磺酸钠,三步综合收率不低于60％。结论：该工艺路线成本较低,操作简单,收率高,适合工业化生产。     

14型肺炎球菌多糖单克隆抗体的制备与初步应用

目的制备14型肺炎球菌多糖单克隆抗体,并进行鉴定及初步应用。方法制备免疫原,免疫BALB/c小鼠,制备单克隆抗体,经3次亚克隆筛选单抗细胞株,并制备腹水。对筛选的单抗进行ELISA效价、特异性、亚型、中和效价、浊度法特异性等鉴定。采用制备的单抗腹水检测市售23价肺炎球菌多糖疫苗、7价肺炎球菌结合疫苗中14型肺炎球菌多糖含量,计算回收率;并与丹麦血清研究所(State Serum Institute,SSI)14型肺炎球菌多糖兔多抗血清检测结果进行比较,计算两者之间的变异系数(CV)。结果共获得7株14型肺炎球菌多糖单抗细胞株:PP-14-101~107,效价分别为105、104、104、104、105、105、105;7株单抗与23价肺炎疫苗中包含的除14型以外的其他所有血清型的多糖均不发生交叉反应;PP-14-101、104、105、106这4株单抗具有免疫浊度趋势,另3株单抗无浊度信号;PP-14-101和PP-14-104株单抗与6A、33F型肺炎球菌多糖具有一定的交叉反应,而PP-14-105和PP-14-106株单抗与23型多糖均无交叉反应;PP-14-101、PP-14-104株单抗为Ig M亚型,PP-14-105、PP-14-106株单抗为Ig G1亚型,PP-14-106比PP-14-105株单抗稀释倍数高,反应性更强,因此选择PP-14-106株单抗为目标单抗;PP-14-106株单抗腹水对14型肺炎球菌的中和效价远高于1∶17 496,中和效价较高;采用PP-14-106株单抗腹水检测市售23价肺炎球菌多糖疫苗和7价肺炎球菌结合疫苗,回收率为90.2%~120.0%,该单抗与SSI血清检测上述疫苗成品14型肺炎球菌多糖含量的CV值在2.5%~11.9%之间。结论成功获得1株效价高、具有杀菌活性、特异性好的单抗,该单抗具有免疫浊度趋势,能准确测定肺炎球菌疫苗中的多糖含量,可替代SSI血清用于多糖疫苗和结合疫苗中14型肺炎球菌多糖含量的定量检测。     

免疫淀粉微球的制备及其结合特性

研究反相微乳法合成淀粉微球,采用环氧氯丙烷活化淀粉微球,考察了活化过程中的影响因素,确定了活化的最佳优化条件;再将制备的肝特异性脂蛋白(liver specific lipoprotein,LSP)抗体与淀粉微球交联,得到免疫淀粉微球;阐述了免疫淀粉微球对抗原肝特异性脂蛋白(LSP)的结合特性。经测定单位淀粉微球交联LSP抗体的量为7.7 mg/g,单位免疫淀粉微球结合LSP的量为0.5 mg/g,表明被交联的抗体保持了较高的生物活性。     

抗荧光素单克隆抗体免疫磁珠的制备及初步应用

目的制备抗荧光素单克隆抗体免疫磁珠,并进行初步应用。方法采用B淋巴细胞杂交瘤技术制备抗荧光素单克隆抗体;制备腹水,经50%硫酸铵粗提后,再经阴离子交换树脂DE52进一步纯化单克隆抗体;采用氧化共沉淀法制备纳米磁性粒子;乳液聚合法制备羧基磁性微球;用碳二亚胺将抗荧光素单克隆抗体共价偶联于磁性微球表面,制备抗荧光素单克隆抗体免疫磁珠。用制备的免疫磁珠检测乙肝表面抗原,并与市售ELISA试剂盒的检测结果进行比较。结果共获得5株杂交瘤细胞株,其中1F12细胞株分泌的抗体效价、相对亲和力较高,特异性较好;纯化的1F12株单抗的纯度达95%,蛋白含量为2.4 mg/ml,ELISA效价为106,相对亲和力为0.2 mg/L,与FITC标记的BSA可特异性结合;纳米磁性粒子的平均粒径为150 nm,铁含量为71.63%;羧基磁性微球的平均粒径为210 nm,羧基含量约为2.15 mmol/g;每克羧基磁性微球可结合抗荧光素单克隆抗体约12 mg,制备的免疫磁珠可有效结合荧光素标记的蛋白。应用抗荧光素单克隆抗体免疫磁珠检测乙型肝炎表面抗原的灵敏度高于ELISA试剂,检测限达0.1 ng/ml。结论成功制备了抗荧光素单克隆抗体免疫磁珠,其具有应用于免疫检测分析的价值。     

氯化亚铜合成及精制

叙述了氯化亚铜生产方法和精制技术。     

Synthesis and application of streptavidin functionalized organosilica microparticles

This article reports on the chemical synthesis, functionalization and application of streptavidin (SA)‐coated organosilica microparticles. Thiol‐functionalized organosilica microparticles are synthesized via a two‐step approach involving acid‐catalyzed hydrolysis and condensation of 3‐mercaptopropyl trimethoxysilane (MPS), followed by base‐catalyzed condensation. The surfaces of these particles are modified with 3‐aminopropyltriethoxysilane (APS), and characterized by FTIR, XPS, and TGA. Then, APS‐functionalized microparticles are covalently coated with SAs by using glutaraldehyde as a coupling agent, and characterized by FTIR, SEM, and AFM. Immobilization of biotinylated λ DNA onto microparticles via SA‐biotin immuoreaction is confirmed by confocal microscopy. The results show that these SA‐functionalized organosilica microparticles are useful carriers for DNA manipulation at the single molecule level. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41560.     

磁性微球的制备

在总结近年来国内外有关磁性微球研究成果的基础上,对磁性微球进行了分类,着重对高分子磁性微球的几种主要制备方法和工艺过程进行了概述.     

Preparation and characterization of micron‐sized magnetic microspheres by one‐step suspension polymerization

Micron‐sized magnetic microspheres with different functional groups were prepared by one‐step suspension copolymerization of styrene, divinyl benzene, and methyl methacrylate in the presence of oleic acid‐coated magnetic nanoparticles. In the present work, we used benzoyl peroxide as an initiator and poly(vinyl alcohol) (PVA‐1788; degree of polymerization: 1,700; degree of hydrolysis: 88%) as a stabilizer. We also added acrylamide (AM) monomer in the aqueous phase and methacrylic acid (MAA) in the oil phase. The morphology and properties of the resulting magnetic microspheres were examined by optical micrographs (OMs), vibrating‐sample magnetometer (VSM), and Fourier transform infrared spectrometer (FT IR). The results showed the three products have uniform and spherical form with superparamagnetism and well dispersion. Moreover, we found that monomer AM had a little contribution to the copolymerization, and MAA could strikingly decrease the diameter of the final microspheres. The magnetic microspheres with functional groups could be linked well with the IgG‐FITC. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

磁性壳聚糖微球的制备及其对2，4-二氯苯酚的吸附

采用反相悬浮聚合法合成了磁性壳聚糖微球,平均粒径不超过5μm。将其用于2,4-二氯苯酚的吸附研究。考察了吸附时间、吸附温度、DCP浓度以及磁性壳聚糖用量等因素对氯酚吸附的影响。结果表明,温度对吸附影响不大,最佳吸附时间为2h时,吸附剂磁性壳聚糖微球用量为1g。对DCP的吸附率在80％以上。在最佳条件下的吸附容量为1．70mg／g。     

聚丙烯原料净化技术及其工业应用

评述了本体法聚丙烯原料丙烯中水、氧、硫和砷等杂质对聚合的影响以及其净化工艺技术,并介绍了昆山市精细化工研究所开发的丙烯精制催化剂及其工业应用实例。