利用鸡蛋特异性抗体IgY的免疫亲和色谱

〖摘要：以牛血清白蛋白（BSA）作为抗原,利用偶联上BSA的Sepharose-4B的亲和色谱材料从鸡蛋蛋黄中一步分离特异性抗BSA抗体。经SDS-PAGE、双向免疫扩散检验,所洗脱的样品为电泳纯的特异性抗体。反之,将所得抗体再偶联到POROSHY上可分离抗原。同时考察了母鸡免疫过程中特异性抗体随时间的变化趋势。-ZY/摘要〗〖WX/文献〗1Scouten.W.H.AffinityChromatography,bioselectiveadsorptiononinertmatrices.NewYork:Wi     

抗体纯化中亲和色谱配体的研究进展

亲和色谱是用于抗体纯化的有效方法,本文对亲和色谱配体的研究进展进行了综述.     

PerfusionG蛋白亲和色谱测定人体生长激素与抗体免疫反应的活性和摩尔配比

 首次报道了应用ＰｅｒｆｕｓｉｏｎＧ蛋白亲和色谱法快速测定人体生长激素与抗体免疫反应活性和摩尔配比的方法。这一新方法与传统的免疫测试技术相比较，具有快速、易于自动化操作和低劳动强度的优点。     

PerfusionG蛋白亲和色谱测定人体生长激素与抗体免疫反应的活性和摩尔配比

首次报道了应用ＰｅｒｆｕｓｉｏｎＧ蛋白亲和色谱法快速测定人体生长激素与抗体免疫反应活性和摩尔配比的方法。这一新方法与传统的免疫测试技术相比较，具有快速、易于自动化操作和低劳动强度的优点。     

分离尿激酶的亲和色谱填料的制备

 合成了分别以 Sepharose和聚甲基丙烯酸环氧丙酯为基质、对氨基苯甲脒为配基的分离尿激酶的两种亲和色谱填料 ,并用于尿激酶粗品的直接纯化 ,活性回收率分别为 1 0 8.3 %和 43 .4% ,比活提高倍数分别为 9.0 6倍和 3 6.9倍。     

亲和膜色谱

亲和膜色谱又称亲和膜分离，其在蛋白质的分离纯化中作为一种综合性的技术出现在80年代末。亲和膜色谱主要优点是克服了颗粒状多孔载体扩散传质阻力大的缺点，代之以对流传质，这样就可以在较低的操作压和较高的流速下对目标蛋白进行快速的分离和纯化，从而缩短操作时间、提高纯化效率。本文将就近年来亲和膜色谱及其在蛋白质分离和纯化中的应用作一综述性介绍。     

嵌段共聚温敏亲和色谱固定相的制备及其对抗体的分离纯化

抗体药物在癌症治疗和免疫诊断中起着重要作用,但抗体的分离纯化通常采用酸性洗脱,易导致抗体聚集失活等问题。本研究以硅胶为基质,以温敏嵌段聚合物聚[(N-异丙基丙烯酰胺)-b-4-乙烯基吡啶](P[NIPAM-b-4VP])为间隔臂,以4-巯基乙基吡啶(MEP)为配基,制备了一种嵌段共聚温敏亲和色谱固定相SiO₂-P[NIPAM-b-4VP]-MEP,并以牛血清白蛋白(BSA)和γ-球蛋白为模型蛋白,对制备的温敏亲和色谱固定相的色谱性能进行了表征。分别考察了流动相盐浓度和温度对二者分离性能的影响。结果表明,在40℃时该固定相只选择性保留γ-球蛋白,而不保留BSA;在5℃时采用含0.6 mol/L NaCl的Tris-HCl(pH 8.0)缓冲溶液进行洗脱,γ-球蛋白的质量回收率为92.7%。该固定相对γ-球蛋白的吸附量为(71.5±2.1) mg/g(n=3),是传统温敏亲和色谱固定相SiO₂-PNIPAM-MEP的2倍。将该固定相用于人血清中IgG的分离纯化,仅通过改变流动相温度和盐浓度即可一步实现对抗体的分离纯化,纯度大于97.4%±0.7%...     

分离尿激酶的胍基型亲和色谱填料研究

尿激酶的精制一般使用亲和色谱法［’－‘ｊ．目前实际使用和文献报道最多的填料是在 ＳｅＰｈａｒｏｓｅ上键合对氨基苯眯（ｐ－ＡＢＺ）制得的［‘ｊ．这种填料分高效果较好,但机械强度不高,只能用于常压色谱,而且寿命较短．前文［‘１报道了用 ＳｅＰｈａｒｏｓｅ和聚甲基丙烯酸环氧丙酯微球为基质, Ｐ－ＡＢＺ为配基分离尿激酶的亲和色谱填料的对照研究．本文报道以含肥基的有机小分子为配基,ＳｅＰｈａｒｏｓｅ及两种聚甲基丙烯酸环氧丙酯微球为基质的分离尿激酶色谱填料的合成和性能试验．发现肥基己酸和精氨酸为配基的亲和色谱…     

亲和膜色谱技术研究进展

对一种崭新的生物大分子分离技术－－亲和膜色谱技术做了全面的评述,介绍了亲和膜色谱分离的过程,评价了亲和膜基质材料活化、改性方法、给出了配基间隔臂的选择原则,概括了亲和膜色谱理论研究的进展,并对亲和膜色谱技术进行了展望。     

亲和分离在蛋白质泛素化修饰研究中的应用进展

蛋白质泛素化是真核生物最普遍、最复杂的翻译后修饰方式之一,在细胞的信号转导、生长、发育、代谢等生命过程中发挥着重要作用。泛素化过程的失调则与神经退行性疾病、炎症反应、癌症等重大疾病的发生发展密切相关。分析和研究蛋白质泛素化的结构与功能,可望为认识生命、探索疾病调控内在规律和发现新的诊断策略提供重要信息。生命体系的高度复杂性,泛素化修饰位点、结构类型的多变和多样性,时空动态变化等特点给蛋白质泛素化分析研究带来了巨大的挑战。亲和分离以其高选择性成为泛素化蛋白质结构与功能研究的有力工具。免疫亲和分离法基于抗原-抗体相互作用,是最为经典的分离分析方法,已广泛应用于泛素化蛋白质或肽段的富集分离。源于天然泛素受体的泛素结合结构域(ubiquitin binding domains, UBDs)可与泛素或多聚泛素链相互作用。UBDs和基于此发展起来的串联泛素结合实体(tandem ubiquitin-binding entities, TUBEs)已成为蛋白质泛素化功能研究的热门识别分子。各种多肽类化合物的发展也为蛋白质泛素化的结构和功能解析提供新工具。此外,多种亲和识别配基的联合使用,在蛋白质泛素化修饰的高特异性、高灵敏度分析中展现了独特的优势,为认识生命体内的泛素化修饰提供了重要保障。该文对亲和分离方法在蛋白质泛素化修饰分析中的应用及进展进行了综述。     

High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G

High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.     

Enhanced binding by dextran‐grafting to Protein A affinity chromatographic media

Dextran‐grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose‐based matrix, followed by epoxy‐activation and Protein A coupling site‐directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran‐grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction‐dried gel, increased by 24% compared with that of the non‐grafted medium. The binding capacity of dextran‐grafted medium decreased about 7% after 40 cleaning‐in‐place cycles, much lower than that of the non‐grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran‐grafted medium faster than that of non‐grafted one. Atomic force microscopy showed that this dextran‐grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non‐grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high‐performance dextran‐grafted Protein A affinity chromatographic medium has promising applications in large‐scale antibody purification.     

Predictive chromatographic simulations for the optimization of recovery and aggregate clearance during the capture of monoclonal antibodies

Predictive chromatographic simulations were used to assess whether significant aggregate clearance, in addition to high step recovery and limited eluate pool volumes, can be achieved during protein A affinity chromatography capture steps. Such aggregates of the antibody monomer are commonly found in manufacturing processes. A lumped desorption-kinetic limiting model was used to describe the elution from the chromatography column, as batch isotherm measurements indicated no adsorption under elution conditions. In order to quantify the trade-off between step recovery and aggregate clearance, independent experiments were first performed to obtain the key kinetic parameters. These parameters were used in simulations to predict the behavior of bench-scale protein A column runs and identify robust operating windows within which good yields and significant aggregate clearance can be achieved. Two examples are described. For antibody A, a robust window of operation was identified. In this case, the optimal conditions were transferred to pilot-plant scale, and the resulting experimental data were shown to be in good agreement with model predictions. For antibody B, it was found that conditions resulting in high recovery and good aggregate clearance were not robust: at the optimal elution conditions, changes of ±0.1 units in pH or ±1 mS/cm in conductivity affected the results substantially.     

Evaluation of a glucose sensing antibody using weak affinity chromatography

Continuous monitoring of drug levels and endogenous molecules in biological fluids is a developing research area with many applications. One example is the need to improve life for millions of diabetes mellitus patients by continuously monitoring the glucose level. In order to have a dynamic response, the recognition molecule in a continuous sensor should preferentially have a fast dissociation rate and a dissociation constant in the millimolar range. We have evaluated the monoclonal antibody (mAb) 3F1E8-A2 for its potential to be used in a future glucose sensor application. The mAb was generated from hybridomas by immunizing mice with 10 kDa dextran (an alpha1,6-glucose polymer) with the aim of obtaining mAbs that can recognize the glucose monomer. The mAb was immobilized to macroporous silica and the interaction with dextran-derived oligosaccharides was evaluated with weak affinity chromatography (WAC). To measure the low affinities between the mAb 3F1E8-A2 and different monosaccharides, a competitive weak affinity chromatography approach was employed. It was found that the mAb had a higher specificity for glucose compared with other monosaccharides and the dissociation constant (K(d)) towards glucose was determined as 18.8 +/- 2.6 mm.     

重组蛋白A亲和填料的制备及其性能评价

为了获得一种优良的抗体纯化介质,制备了重组金黄色葡萄球菌蛋白A(rProtein A)亲和填料,并考察了所制备的亲和填料的纯化性能。利用自行构建的rProtein A工程菌,经诱导表达、纯化获得rProtein A纯品,将其偶联到经环氧氯丙烷活化的Sepharose 4 Fast Flow凝胶上,得到rProtein A亲和填料,并使用兔抗尿酸氧化酶抗体对该填料的性能进行验证。结果显示,在自制的rProtein A亲和填料上rProtein A浓度为1.5×10~4 mol/L。采用Scatchard模型分析,得到其解离常数和最大表观吸附量分别为2.28×10~7 mol/L和20.697 g/L,说明制得的rProtein A亲和填料对抗体有很好的结合能力。将该填料于0.1 mol/L NaOH溶液中浸泡1 h,其色谱性能未见变化。将该填料用于纯化兔抗体,湿胶结合抗体量可达19 mg/mL;一步柱色谱即可得到电泳纯度的抗体样品,回收率高于96%。本研究为rProtein A亲和填料的国产化奠定了基础。     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

一种凝血酶亲和色谱介质的制备方法

对凝血酶-琼脂糖亲和色谱介质的制备方法进行了研究。首先使用凝血酶和溴化氰活化的琼脂糖制备凝血酶-琼脂糖亲和色谱介质,然后用生色底物法考察亲和色谱介质上凝血酶的活性,以凝血酶活性为指标对最佳偶联条件进行了优化。结果表明最佳条件为使用pH 8.3的Na₂CO₃-NaHCO₃溶液(含0.5 mol/L NaCl)为缓冲溶液,凝血酶用量为每1 g色谱介质加入凝血酶200 U,室温反应10 h。在最佳条件下所制备的色谱介质有较好的稳定性,在4℃条件下存放40天,亲和介质上的凝血酶活性仍有70.6%保留。该亲和色谱介质可广泛用于含凝血酶抑制剂的天然药物筛选和分离纯化。     

Application of mixed mode resins for the purification of antibodies

The downstream processing of monoclonal antibodies from cell culture supernatant is usually done by a number of chromatographic and non-chromatographic steps. Efforts are taken to reduce the costs associated to those steps, while maintaining a high product purity. A possibility to reach this goal is the reduction of the number of chromatographic steps using mixed mode resins that offer more than one functionality in one chromatographic step. In this work, a commercially available mixed mode resin was evaluated systematically with respect to the adsorption of proteins. The Henry coefficient, which quantifies the adsorption strength, was measured for the full working range of the stationary phase as a function of the salt concentration and the pH. The results were compared to a conventional anion exchange and a hydrophobic interaction resin. Furthermore, the resin was applied for the polishing step of an antibody from an industrial clarified cell culture supernatant.     

Binding and elution strategy for improved performance of arginine affinity chromatography in supercoiled plasmid DNA purification

New interesting strategies for plasmid DNA (pDNA) purification were designed, exploiting affinity interactions between amino acids and nucleic acids. The potential application of arginine-based chromatography to purify pDNA has been recently described in our work; however, to achieve higher efficiency and selectivity in arginine affinity chromatography, it is essential to characterize the behaviour of binding/elution of supercoiled (sc) isoforms. In this study, two different strategies based on increased sodium chloride (225-250 mm) or arginine (20-70 mm) stepwise gradients are described to purify sc isoforms. Thus, it was proved that well-defined binding/elution conditions are crucial to enhance the purification performance, resulting in an improvement of the final plasmids yields and transfection efficiency, as this could represent a significant impact on therapeutic applications of the purified sc isoform.