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ProteinA亲合膜色谱柱的性能及从人血浆中纯化免疫球蛋白的研究 总被引:5,自引:3,他引:5
以木纤维为基质,与甲基丙烯酸环氧丙酯共聚接枝合成了复合膜介质,用复合膜介质制备了proteinA亲合膜色谱柱,考察了proteinA亲合膜色谱柱液相流动特性和吸附性能。实验证明:流速与亲合膜色谱柱柱压呈线性关系,当流速为3mL/min时,柱压为160kPa。免疫球蛋白(IgG)浓度和上样速度是影响proteinA键合容量的重要因素,对其进行了优化研究。用动态吸附法确定了对人IgG动态最大吸附能力可达21.7mg/g(干介质)。 相似文献
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甲基丙烯酸缩水甘油酯(GMA)为单体,二乙烯基苯(DVB)和三聚异氰尿酸三烯丙酯(TAIC)为交联剂,在致孔剂甲苯和正庚烷存在下,直接以Φ4.6×100mm色谱柱管为模具,通过原位聚合制备了聚(甲基丙烯酸缩水甘油酯-二乙烯基苯-三聚异氰尿酸三烯丙酯)(PGDT)型连续床.然后,利用二乙胺和骨架结构中的环氧基反应得到阴离子交换型连续床.对连续床的化学结构、孔结构及其对蛋白质的分离性能进行了研究.实验结果表明,连续床内部含有大量类似渠道的大孔,孔径为1~2μm.在流速高达3250cm/h时,背压仅为9.89Mpa.而且流速对色谱分离效率的影响小,高流速下仍能得到高分离效率,可以通过提高流速实现蛋白质的快速分离. 相似文献
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接枝聚硅氧烷环糊精固定相溶胶-凝胶毛细管气相色谱柱分离内酯对映体 总被引:1,自引:0,他引:1
参照文献方法合成了4种以环糊精为侧链的含氢聚甲基硅氧烷。在4种不同的合成物中,环糊精或是以本身不同的位置与主链相连,或是连接臂的结构不同,亦或是连接臂的长短各异。分别用合成的4种化合物为固定相,首次采用溶胶-凝胶法制备了气相色谱手性毛细管柱。在这些色谱柱上,以手性化合物(±)γ-苯基-γ-丁内酯为探测物对4种色谱柱的柱效率、手性选择性、容量因子、分离度及固定相的极性等各种色谱参数进行了测定,并将测定的数值进行了比较。发现环糊精以圆台较小的一端与含氢硅油相连,连接臂中不含苯氧基而含有醚键,连接臂较短(含4个亚甲基)的固定相是所合成的4种固定相中的最佳选择。 相似文献
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非电荷型毛细管电色谱原位柱的色谱行为研究──阴离子表面活性剂的动态改性 总被引:7,自引:5,他引:2
采用原位聚合的方法在毛细管中合成了非电荷型连续床电色谱原位柱 ,通过在电色谱流动相中加入阴离子表面活性剂十二烷基硫酸钠 (SDS)进行动态改性使其产生电渗流 ,考察了SDS浓度及有机改性剂浓度等因素对电渗流的影响。此类连续床柱制备容易 ,柱效可达 14万理论塔板 /m ,在不同的操作条件下有良好的稳定性 ,连续 10次运行 ,其死时间t0 与保留时间的精密度分别为 0 .2 2 %和 <0 .5 6 %。 相似文献
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以环氧活化聚甲基丙烯酸酯大孔微球(FastSep-epoxy)为基质,通过接枝聚(烯丙基胺)(PAA)制备了大孔阴离子交换色谱介质(FastSep-PAA)。考察了介质的合成条件对离子交换容量(IC)及蛋白结合容量的影响,发现IC随PAA浓度、反应时间和溶液pH的增加均表现为增长趋势;同时结合蛋白吸附容量的变化选择了最优合成条件。通过扫描电子显微镜观察介质的表面形貌,发现其孔隙连通性较好,且接枝前后介质孔道结构无明显变化,PAA配基密度对介质结构无明显影响。此外,通过压汞法和氮气吸附法测定接枝前后不同介质的孔径尺寸和孔径分布情况,并考察该类介质的孔径与蛋白吸附行为的关系,发现其蛋白结合容量未出现随介质孔径尺寸增加而显著下降的现象,且孔径尺寸增加更有利于蛋白在介质内部传质。在126 cm/h的流速下FastSep-PAA介质的原始孔径(即FastSep-epoxy的孔径)为400 nm时的蛋白动态结合容量(DBC)最高(70.3 g/L),该孔径下介质比表面积大,蛋白可吸附位点较多;原始孔径为700 nm及以下的介质蛋白DBC均随流速增加而均有一定下降;原始孔径为1 000 nm的介质蛋... 相似文献
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切流膜色谱柱是免疫吸附治疗中实现全血灌流的一种新的模式。实验研究表明,切流膜色谱柱的结构和血液流动形式对血细胞的损害很小。分别考察了水、血浆、血液等不同流体流速与切流膜色谱柱柱压降之间的关系,柱压降随着流体流速和粘度的升高而增高,血液流速为120mL/min时,柱压降达到93kPa;考察了ProteinA切流膜色谱柱对人血浆中免疫球蛋白IgG的吸附能力,切流膜色谱柱ProteinA健合量为139mg(6mgProteinA/g干介质),血浆在柱中循环1h可吸附IgG553mg(23.8mgIgG/g平介质),而血液在柱中循环1h,可吸附IgG499.4mm(21.5msIgG/g卡介质)。 相似文献
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人免疫球蛋白 G(HIg G)是一种重要的生物大分子 ,是人血浆中的主要成分之一 ,通常采用免疫学的方法测定 .蛋白 A(Protein A)与免疫球蛋白 (HIg G)的 Fc区之间具有很强的特异性亲和作用 ,因而固载蛋白 A的亲和介质可用于免疫球蛋白及单克隆抗体的分离、纯化和分析测定[1~ 3 ] .根据固定相存在形式的不同 ,毛细管色谱柱主要有开管、填充和连续床柱 3种方式 .连续床具有相比高、易制备 (一步合成 )、孔径易控制、不需烧塞子和易改性等优点 .连续床与其它常用的亲和介质 (如球型凝胶颗粒、灌流色谱基质 [4、 5] 、膜介质 [6,7] 等 )相比具… 相似文献
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蛋白A高效亲和膜色谱法测定人血浆中免疫球蛋白G的含量 总被引:6,自引:1,他引:6
研究了蛋白A高效亲和膜色谱对水溶液及人血浆中人免疫球蛋白G(HIgG)的特异性吸附和定量测定。方法具有较高的精确度和较好的重复性:HIgG标样5次重复进样的相对标准偏差为1.5%,人血浆样品3次重复进样的相对标准偏差为3.6%;所测得的定量标定曲线的线性相关系数达到0.9993;不含己二胺间隔臂的亲和介质的非特异性吸附极低,基本检测不出来。快速实验中,一次分析可在0.5min内完成。实验表明,利用所建立的方法对人血浆中的HIgG进行定量测定可以得到较为满意的结果。 相似文献
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This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%. 相似文献
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2‐Methacrylamidohistidine (MAH) as a pseudospecific ligand was synthesized from methacryl chloride and histidine. Spherical beads with an average size of 50–63 μm were obtained by the radical suspension polymerization of MAH and 2‐hydroxyethyl methacrylate (HEMA) conducted in an aqueous dispersion medium. Owing to the reasonably rough character of the bead surface, P(HEMA‐co‐MAH) beads had a specific surface area of 17.6 m2·g–1. Synthesized MAH was characterized by NMR. P(HEMA‐co‐MAH) beads were characterized by swelling studies, FT‐IR spectroscopy, scanning electron microscopy (SEM) and elemental analysis. P(HEMA‐co‐MAH) affinity beads with a swelling ratio of 65% were used in the separation of human immunoglobulin G (HIgG) from aqueous solutions and human plasma. The maximum HIgG adsorption on the P(HEMA‐co‐MAH) adsorbents was observed at pH 7.4 for phosphate and at pH 6.0 for morpholinoethanesulfonic acid buffers. The HIgG adsorption onto the PHEMA adsorbents was negligible. Higher adsorption values (up to 46.5 mg·g–1) were obtained when the P(HEMA‐co‐MAH) adsorbents were used in aqueous solutions. Much higher amounts of HIgG were adsorbed from human plasma (up to 73.8 mg·g–1). Adsorption capacities of other blood proteins were obtained as 3.2 mg·g–1 for fibrinogen and 4.6 mg·g–1 for albumin. The total protein adsorption was determined to be 82.2 mg·g–1. The pseudospecific affinity beads allowed one‐step separation of HIgG from human plasma. HIgG molecules could be repeatedly adsorbed and desorbed with these adsorbents without noticeable loss in their HIgG adsorption capacity. 相似文献
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本文发展了一种基于纳米金介导生物沉积铂并以铂催化氢还原伏安法进行检测的高灵敏电化学免疫分析新方法。该方法采用夹心免疫分析模式,实现了人免疫球蛋白(HIgG)的测定。首先在聚苯乙烯微孔板中固定羊抗HIgG捕获抗体,HIgG捕获后,碱性磷酸酶标记的HIgG抗体修饰的纳米金探针通过与HIgG的形成的夹心复合物而结合在微孔板上。结合的碱性磷酸酶催化抗坏血酸磷酸酯底物水解产生抗坏血酸,后者在纳米金上介导下还原铂离子沉积于纳米金表面。沉积的金属铂用王水溶解并电富集于玻碳电极上。通过测定铂催化氢还原产生的阴极电流,可实现HIgG的高灵敏分析。催化氢还原电流与HIgG浓度对数在0.1~100ng/ml之间呈线性相关性,检测限达22pg/ml。由于铂催化氢还原的高灵敏度及纳米金介导的生物沉积放大反应,该法具有较高的分析灵敏度,且免疫分析微孔板模式使得该法可同时用于大量样品的分析。 相似文献
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We report a field effect transistor (FET) based on a network of single-walled carbon nanotubes (SWCNTs) which can selectively detect human immunoglobulin G (HIgG). HIgG antibodies, which are strongly adsorbed onto the walls of the SWCNTs, are the basic elements of the recognition layer. The non-specific binding of proteins and the effects of other interferences are avoided by covering the non-adsorbed areas of the SWCNTs with Tween 20. The selectivity of the sensor has been tested against bovine serum albumin (BSA), the most abundant protein in plasma. HIgG in aqueous solution with concentrations from 1.25 mg L(-1) (8 nM) can be readily detected with response times of about 10 min. The SWCNT networks that form the basis of the sensor are easily grown by chemical vapour deposition. Silver screen-printed electrodes make the sensor quick to build. The sensitivity obtained with this sensor is similar to other FET devices based on SWCNTs built using much more complicated lithography processes. Moreover, the sensor is a reagentless device that does not need labels to detect HIgG. 相似文献
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Ke-Jing Huang De-Jun Niu Jun-Yong Sun Xiao-Li Zhu Jun-Jie Zhu 《Analytical and bioanalytical chemistry》2010,397(8):3553-3561
A novel experimental methodology based on a Prussian blue (PB) and gold nanoparticles (AuNPs) modified carbon ionic liquid
electrode (CILE) was developed for use in a label-free amperometric immunosensor for the sensitive detection of human immunoglobulin
G (HIgG) as a model protein. The CILE was fabricated by using the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate
as binder. Controllable electrodeposition of PB on the surface of the CILE and coating with 3-aminopropyl triethylene silane
(APS) formed a film with high electronic catalytic activity and large surface area for the assembly of AuNPs and further immobilization
of HIgG antibody. The electrochemistry of the formed nanocomposite biofilm was investigated by electrochemical techniques
including cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. The HIgG concentration
was measured through the decrease of amperometric responses in the corresponding specific binding of antigen and antibody.
The decreased differential pulse voltammetric values were proportional to the HIgG concentration in two ranges, 0.05–1.25 ng mL−1 and 1.25–40 ng mL−1, with a detection limit of 0.001 ng mL−1 (S/N = 3). This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of
the AuNPs, ionic liquid, and PB amplified electrochemical detection and would therefore be valuable for clinical immunoassays. 相似文献
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Ito S Imura T Fukuoka T Morita T Sakai H Abe M Kitamoto D 《Colloids and surfaces. B, Biointerfaces》2007,58(2):165-171
Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (Kd = 1.7 × 10−6 M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (Kd = 2.4 × 10−7 M), and HIgM (Kd = 2.2 × 10−7 M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (ka1, ka2) increase with an increase in the number of antibody binding sites, while those for dissociation (kd1, kd2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins. 相似文献
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HE Wenying YAO Xiaojun LIU Pengjun GAO Zhenxia & HU Zhide . Department of Chemistry Lanzhou University Lanzhou China . Department of Chemistry Hainan Normal University Hainan China 《中国科学B辑(英文版)》2006,49(6):550-559
The fluorogenic property of guaiacol was exploited for the first time to analyze the interaction with target protein as a probe by molecular modeling, fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. Molecular docking was performed to reveal the possible binding mode or mechanism and suggested that guaiacol can strongly bind to human immu- noglobulin (HIgG). It is considered that guaiacol binds to HIgG mainly by a hydrophobic interaction and there are two hydrogen bond interactions between the drug and the residues LEU 80 and ASP 65, which is in good agreement with the results from the experimental thermodynamic parameters (the enthalpy change △H0 and the entropy change △S0 were calculated to be 65.55 kJ·mol-1 and 132.95 J·mol-1·K-1 according to the Vant’ Hoff equation). Data obtained by the fluorescence spectroscopy indicated that binding of guaiacol with HIgG leads to dramatic enhancement in the fluorescence emission intensity along with significant occurrence of efficient Frster resonance energy transfer (FRET) from the residue of HIgG to the protein bound guaiacol. From the low value of fluorescence anisotropy (r = 0.06), it is argued that the probe molecule is located in the motionally unrestricted environment of the protein. The alterations of protein’s secondary structure in the presence of guaiacol in aqueous solution were quantitatively calculated by the evidences from FT-IR and CD spectroscopes. 相似文献
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A sensitive, highly specific immunoassay method has been developed by measuring the enhanced resonance light scattering (RLS) signals of immunoreactions with simultaneously scanning both the excitation and the emission monochromators of a common spectrofluorometer. For a given content of antibody (Ab), the RLS signals of an immunoreaction follow Gaussian distribution with antigen (Ag) concentration. The central position of the Gaussian curve represents the concentration of given Ab, and the half bandwidth has proved to be a characteristic constant of a given Ab-Ag immunoreaction. With the RLS signals, the limit of detection for human immunoglobulin G (HIgG) in serum samples could reach 10 ng ml−1, and the concentration of HIgG in blood serum samples could be detected with the recovery of 90.2-107.7% and R.S.D. of 0.8-2.7%. The results of determination for three human serum samples are identical to those obtained by immunoturbidimetry. 相似文献