白蛋白在以染料为配基的醋酸纤维滤棒上的等温吸附行为(英文)

 以醋酸纤维滤棒为基质 ,染料CibacronBlueF3GA为配基 ,合成了一种新的染料亲和介质 ;分别以牛血清白蛋白 (BSA)和人血清白蛋白 (HSA)为对象 ,用静态法进行了吸附实验 ,得到了相应的亲和等温吸附曲线 ;对曲线按Langmuir模型和Freundlich模型分别进行拟合 ;结果表明 ,醋酸纤维滤棒染料亲和介质对BSA和HSA的等温吸附遵循Freundlich模型。采用该亲和介质装柱并分离实际样品人血浆 ,可得到纯化的人血清白蛋白。     

基于免疫磁分离的荧光微球免疫层析法检测猪霍乱沙门氏菌

建立了基于免疫磁分离的荧光微球免疫层析法,检测猪霍乱沙门氏菌.待检样品经免疫磁分离富集和热洗脱处理后,用荧光微球免疫层析试纸条进行检测.每毫克纳米磁珠标记30μg抗体制备的免疫磁珠,对浓度为102 ～ 106 CFU/mL的猪霍乱沙门氏菌的捕获率均大于90％,特异性好;在pH=6时,以300μ,g/mg猪霍乱沙门氏菌单抗11D8-D4标记荧光微球,制备免疫荧光微球;以2.0 mg/mL猪霍乱沙门氏菌单抗5F11-B11喷涂检测线(T线),以1.0 mg/mL驴抗鼠IgG喷涂质控线(C线),制备免疫层析试纸条.采用建立的基于免疫磁分离的荧光微球免疫层析方法检测猪霍乱沙门氏菌,在PBS缓冲液中检出限为1.5×105 CFU/mL,牛奶中检出限为7.6×105 CFU/mL,与直接采用荧光微球免疫层析方法检测相比,检出限分别降低了10倍和200倍.本方法可有效富集牛奶中的沙门氏菌,避免了基质干扰,灵敏度大大提高,具有较好的应用前景.     

免疫质谱法检测肝癌血清多肽标志物

建立免疫亲和富集分离与质谱分析相结合的免疫质谱法用于血清多肽标志物pep5的检测。多克隆抗体与蛋白A琼脂糖颗粒偶联用于捕获pep5,基质辅助激光解析电离飞行时间质谱检测标志物。本方法的线性范围为1～48 nmol/L,检出限0.2 nmol/L;回收率为93.0%～103.1%,相对标准偏差(RSD)为6.4%～11.8%;反复冻融3次pep5,检测结果稳定;检测临床肝癌/正常血清样本灵敏度为78.0%、特异度为90%。该方法方便、快速、准确,适用于临床样本中低浓度标志物的检测研究,对于癌症的早期诊断具有重要意义。     

新型花菁染料-蛋白质散射体系的研究与应用

基于蛋白质对花菁染料的共振散射效应,以新型水溶性碳菁染料(1,1′-丙磺酸-3,3,3′,3′-四甲基吲哚三次甲基碳菁-5,5′-二磺酸钾)为探针,建立了一种新的蛋白质共振散射检测体系.在最优条件下,蛋白质对该碳菁染料具有明显的散射增强作用,散射强度与蛋白质浓度成良好的线性关系,牛血清蛋白BSA和人血清蛋白HSA线性响应范围分别为0.20～15.0μg/mL和0.50～10.0μg/mL,检测灵敏度(3σ/K)为0.05、0.10μg/mL.测定了BSA血清合成样品,回收率94.5%～104.0%;测定HSA血清合成样品,回收率95.6%～103.0%,测定相对标准偏差为1.1%～1.9%,测定较为稳定、准确.     

基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。     

基于适配体的实时定量PCR检测血清中的人促红细胞生成素

基于适配体的高特异识别能力和可扩增性,构建了经磁分离后实时定量PCR检测重组人促红细胞生成素-α(rHuEPO -α)的新方法;针对实际血清样品中高丰度蛋白质等的干扰,利用碱基互补配对原理设计合成了分别与适配体两端引物区结合的互补链,通过凝胶迁移阻滞分析(EMSA)筛选出最佳的互补链,并将生物素化的互补链连接到链霉亲和素磁珠上,以此为探针捕获复杂基质中形成的待测复合物.研究结果表明,结合该样品前处理策略,该方法可成功地应用于正常人血清中的rHuEPO -α定量检测,检出限为25pmol/L,线性范围为50 pmol/L～ 50 nmol/L.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

聚丙烯酸/萘酰亚胺纳米粒荧光增强法测定蛋白质

制备了水溶性的聚丙烯酸/萘酰亚胺荧光纳米粒,并利用牛血清白蛋白(BSA)对其荧光的显著增强效应,建立了测定蛋白质的新方法。在pH=3.0磷酸盐缓冲溶液及室温条件下,该纳米粒与BSA的作用5 min内即完成。以444nm为激发波长,在535nm处测定纳米粒/BSA体系的荧光,其荧光强度增加与0.15~7.5μmol·L-1浓度范围内的BSA呈线性关系,检测限为0.095μmol·L-1。该方法简单、快速、灵敏且重现性好,用于BSA合成样品及牛奶样品中蛋白质的测定,回收率范围为96.7%~102.6%,相对标准偏差(RSD)在3.91%以内。     

氰基乙基-5-氯尿嘧啶核苷探针测定蛋白质的同步荧光法研究

基于核苷类药物中间体氰基乙基-5-氯尿嘧啶(CECU)与血清白蛋白相互作用生成复合物,导致血清白蛋白的内源荧光产生特异性变化,而建立了以氰基乙基-5-氯尿嘧啶为荧光探针,用固定波长同步荧光光谱技术测定人血清白蛋白(HSA)和牛血清白蛋白(BSA)的新方法。结果表明,在最佳实验条件下,体系的荧光强度与人血清白蛋白和牛血清白蛋白分别在2.76~497μg/mL和3.90~507μg/mL范围内呈良好的线性关系,检出限分别为1.64和1.72μg/mL(n=11)。该方法灵敏度高、稳定时间长、选择性好、线性范围宽。方法已用于血清样品中蛋白质的快速测定。     

免疫亲和萃取-超高效液相色谱-串联质谱法分离测定中成药及中药材中的5种黄曲霉毒素

利用免疫亲和萃取结合超高效液相色谱-串联四极杆质谱技术(UHPLC-ESI/QqQ-MS/MS)建立了中成药及中药材中5种黄曲霉毒素(B1、B2、G1、G2和M1)的提取、分离、确证与定量方法。样品经80%(体积分数)的甲醇水溶液提取和免疫亲和固相萃取后,采用UHPLC-ESI/QqQ-MS/MS的多反应监测模式实现分离、鉴定和外标法定量。5种目标毒素标准溶液的检出限(LOD)为0.05～0.3 μg/L。在0.5～100 μg/L的基质添加浓度范围内具有良好的线性关系(r2＞0.99);以甘草为例,当添加水平为1.0 μg/kg和5.0 μg/kg时,得到62.3%～82.4%的回收率(相对标准偏差(RSD)＜10%, n=6)。该方法灵敏度高、选择性和重复性好、回收率较高、检测速度快,适用于中成药及中药材等复杂基体中多种黄曲霉毒素的快速分析与筛查。     

The preparation of bovine serum albumin surface-imprinted superparamagnetic polymer with the assistance of basic functional monomer and its application for protein separation

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Characterization of the Binding Performance of Silica-Based Immunoaffinity Adsorbents Prepared via Online Coupling Reactions

Antibodies against a conjugate of daidzein and bovine serum albumin (BSA) were immobilized onto dihydrazide-activated silica beads via online coupling reactions. The binding performances of the obtained immunoaffinity adsorbents to the corresponding hapten and carrier protein were all optimized and characterized. It was found that online coupling reactions offered a convenient and practical way to generate the requisite immunoaffinity columns. The optimum binding buffers for BSA and daidzein were found to be pH 5.0, 0.01 mol L^?1 phosphate-buffered saline (PBS) and 0.05% Tween-20 in pure water, indicating different interactions between the immobilized antibodies with different parts of the antigen. Under the optimum operating conditions, the silica-based immunoaffinity adsorbents showed specific and selective binding properties to the target protein; while for small molecules, a mixed adsorption mechanism caused by both specific and non-specific interactions was observed. For comparison, offline coupled silica-antibody immunoaffinity adsorbents and Sepharose 4B support immobilized with the same antibody were also prepared and tested. The experimental results of this study may provide useful information for further development of other high-performance immunoaffinity chromatographic methods for different purposes.     

Migration behavior and separation of active components in Glycyrrhiza uralensis Fisch and its commercial extract by micellar electrokinetic capillary chromatography

1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS), 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and 2-(p-toluidino)naphthalene-6-sulfonic acid (2,6-TNS) were evaluated as additives in different buffers for the detection of bovine whey proteins using laser-induced fluorescence (LIF) monitoring in capillary electrophoresis (CE). These N-arylaminonaphthalene sulfonates furnish a large fluorescence emission when associated to some proteins whereas their emission in aqueous buffers, such as those used in CE separations, is very small. To select the best detection conditions, the fluorescence of these probes was first compared using experiments carried out in a fluorescence spectrophotometer. Using bovine serum albumin (BSA) as a model protein, it was demonstrated that 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) buffer (pH 8 and pH 10.2) and the fluorescent probe 2,6-TNS gave rise to the highest increase in fluorescence for BSA. When the composition of these separation buffers was optimized for the electrophoretic separations, CHES buffer, pH 10.2 was chosen as the most suitable buffer to detect bovine whey proteins. The limit of detection obtained for some whey proteins in CE separations was about 6.10(-8) M for BSA, 3.10(-7) M for beta-lactoglobulin A (beta-LGA), 3.10(-7) M for beta-lactoglobulin B (beta-LGB), and 3.10(-6) M for alpha-lactalbumin (alpha-LA). These detection limits were compared to those achieved using UV detection under the same separation conditions. The results showed that the detection limits of BSA, beta-LGA and beta-LGB were twice as good using LIF than with UV detection. However, the limit of detection for alpha-LA was better when UV was used. The applicability of LIF detection to CE separation of whey proteins in bovine milk samples was also demonstrated.     

毛细管等电聚焦和电渗泵驱动聚焦区带分离蛋白质

建立了一种利用电渗泵驱动毛细管内的聚焦区带,实现毛细管电泳等电聚焦分离蛋白质的方法。通过控制电压来调节泵的输出流量,从而调节聚焦区带的迁移速度。适用于毛细管电泳等电聚焦两步法分离蛋白质等两性物质。考察了对牛血清白蛋白和溶菌酶两种粗提蛋白质混合物的分离,迁移时间的RSD分别为1.6%和1.3%,峰面积的RSD均为1.6%,证明方法可行。     

Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.     

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT

A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.     

Band broadening caused by the multiple labeling of proteins in micellar electrokinetic chromatography with diode laser-induced fluorescence detection

When a labeling reagent is used, in the determination of proteins by capillary electrophoresis with laser-induced fluorescence detection, the multiple labeling of proteins frequently occurs, which can degrade the separation efficiency. In order to understand the influence of the multiple labeling of proteins on separation efficiency, the band broadening caused by a labeling reaction between bovine serum albumin (BSA) and a cyanine fluorescent dye (Cy5) was investigated using micellar electrokinetic chromatography in conjunction with diode laser-induced fluorometry. With the aid of an internal standard, methylene blue, the height equivalent to the theoretical plate (HETP) ratio of BSA to methylene blue was used as an indicator for band broadening under optimum separation conditions. Labeling conditions, including reaction buffer pH, reaction time, and initial concentration of Cy5 to bovine serum albumin, were found to influence the HETP ratio. The separation efficiency for the labeled protein was degraded by experimental conditions employed in the labeling, which indicates an increase in the heterogeneity of the final products.     

Plastic microchip liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry using monolithic columns

A prototype array of monolithic liquid chromatography (LC) columns was prepared in a plastic microfluidic device for the off-line interface with matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microfluidic channels were fabricated on a cyclic olefin copolymer (COC) plate by hot embossing. An array of methacrylate monolithic columns was prepared in the microfluidic channels by UV-initiated polymerization. The deposition system employed a pulsed electric field to transfer the effluents from multiple columns directly onto MALDI targets with a non-contact deposition method reported by Ericson et al. [C. Ericson, Q.T. Phung, D.M. Horn, E.C. Peters, J.R. Fitchett, S.B. Ficarro, A.R. Salmon, L.M. Brill, A. Brock, Anal. Chem. 75 (2003) 2309]. To characterize the off-line interface of the multiple-channel microchip LC and the MALDI-MS for the analysis of peptide mixtures, the separation efficiency and reproducibility tests in each column were carried out by separating a peptide mixture from tryptic digested proteins and depositing the multiple effluents simultaneously on the MALDI target plate. Using a MALDI-TOF mass spectrometer with a mass accuracy of +/-1 Da for peptide assignments of digested bovine serum albumin (BSA), amino acid sequence coverage of around 59% was obtained for the microchip LC-MALDI-MS compared to 23% obtained by the MALDI-MS method without LC separation. In sensitivity tests for the detection of low abundance proteins in the presence of high concentration protein mixtures, as low as 10 fmol/mul (S/N = 10) of a spiked peptide in 1 microg of digested BSA could be detected. In the analysis of a mixture of three digested proteins (BSA, myoglobin, and cytochrome c), more than twice the amino acid sequence coverage was obtained for the microchip LC-MALDI-MS compared to MALDI-MS alone.     

Determination of proteins at nanogram levels with Bordeaux red based on the enhancement of resonance light scattering

A simple, sensitive and selective method was proposed for the determination of proteins by using a resonance light scattering technique. The weak resonance light scattering (RLS) of Bordeaux red (BR) can be enhanced greatly in the pH range 3.87-3.96 by the addition of micro amounts of proteins, resulting in four characteristic peaks in the wavelength range 250-600 nm. At the maximal wavelength of 363 nm, the enhanced RLS is proportional to the concentration of proteins in the range 0.12-10.8 microg ml-1 for bovine serum albumin (BSA) and 0.24-18.0 microg ml-1 for human serum albumin (HSA). The detection limits were 40.0 and 52.9 ng ml-1 for BSA and HSA, respectively. The present method has been applied to the determination of total proteins in human serum, urine and saliva samples. The obtained results are in good agreement with those obtained by the Bradford method with relative standard deviations (R.S.D.) of 0.9-2.3%.