国产免疫磁珠与溴化氢活化Sepharose 4B亲和层析分离羊抗人IgG的比较

目的比较国产免疫磁珠与溴化氢活化Sepharose 4B两种不同介质偶联人免疫球蛋白纯化羊抗人IgG的差别。方法采用亲和层析的方法纯化羊抗人IgG,通过血凝实验检测不同介质纯化产物的效价和特异性。通过SDS-PAGE检测其纯度。通过计算偶联率及蛋白回收率,比较两种介质纯化效率。结果两种不同介质纯化的羊抗人IgG特异性良好,效价均为1∶512,SDS-PAGE检测均为单一条带。采用两种介质纯化羊抗人IgG,蛋白回收率分别为10·03%和17·45%。结论两种不同介质采用亲和层析方法纯化均可获得纯度较高的羊抗人IgG。     

链球菌蛋白G免疫球蛋白结合域C3区串联体的表达、筛选与初步应用

筛选并制备高效结合抗体的重组蛋白G亲和层析介质。以链球菌蛋白G C3区为结构单元,拼接形成3、4、5、6个重复C3片段的串联体,分别克隆至PET21,并在E.coli BL21(DE3)中表达。经Ni~(2+)纯化的重组蛋白分别偶联至Purose 4 Fast Flow制备亲和层析介质,比较介质的吸附情况,发现重组四串体对h IgG的吸附量最高,其静态饱和吸附量为66.79 mg×mL~(-1),动态吸附载量为35 mg×mL~(-1)介质,优于同类型商品介质。利用该介质分离纯化小鼠腹水和人血清中的IgG,纯度在95%左右,回收率不低于80%。     

丙酮酸改性壳聚糖对金属离子的吸附性能研究

丙酮酸经Schiff碱反应对壳聚糖进行修饰 ,合成了高取代的水溶性丙酮酸缩壳聚糖 (PCTS) ,研究了PCTS、SCTS(水杨醛改性壳聚糖 )、CTS(壳聚糖 )对Cu(Ⅱ )、Zn(Ⅱ )、Co(Ⅱ )的静态吸附性能 ,并采用正交实验法考察了金属离子浓度、介质酸度、吸附量和吸附时间对吸附剂去除金属离子能力的影响。结果表明 ,PCTS的吸附性能优于SCTS与CTS ,对Cu(Ⅱ )、Zn(Ⅱ )、Co(Ⅱ )的吸附容量 (pH =7 0 )分别为 2 79 56、1 96 63、70 2 1mg/g ,金属离子浓度、介质酸度对吸附性能影响大 ,而吸附剂用量、吸附时间对吸附性能影响较小。     

改性壳聚糖树脂对铜的吸附研究

以壳聚糖为原料,将其进行希夫碱改性,制备改性壳聚糖树脂V-CS,研究其对Cu2+的吸附性能。吸附优化结果表明,Cu2+溶液的初始浓度为100 mg/L,p H=5.0,吸附剂投加量为0.04 g,振荡吸附4 h,吸附量可达62.67 mg/g。可用于水体中Cu2+的富集和分离。     

无助剂下丙烯酸改性壳聚糖对Cu(Ⅱ)的吸附

采用无助剂条件丙烯酸改性壳聚糖制备吸附剂(CTS-AA),深入考察了pH对吸附Cu~(2+)的影响,pH大约为5.35时为最佳,吸附量达到158.9 mg/g,比壳聚糖的吸附量提高了21%,同时发现低温更有利于吸附的进行。     

凹凸棒土/壳聚糖复合物的制备和吸附性能探究

以凹凸棒土为载体，将壳聚糖负载于凹凸棒土，得到凹凸棒土/壳聚糖(AT/CTS)改性吸附剂，用于染料废水的处理。利用紫外光谱、红外光谱和X射线衍射对AT/CTS的结构进行分析，初步探讨了AT/CTS对染料亚甲基蓝吸附性能的影响因素。结果表明：最佳吸附性能出现在吸附剂用量为10 mg、10 mL初始质量浓度70 mg·L^-1的亚甲基蓝溶液、温度30℃、pH值为13和吸附时间30 min时，此时的平均吸附率达到98.5%。同时AT/CTS吸附剂对亚甲基蓝吸附动力学符合准二级动力学模型，吸附过程符合Langmuir等温方程。     

新型改性壳聚糖的制备及其对2,4-二氯苯酚的吸附

采用水杨醛与壳聚糖反应生成Schiff’s碱,再用NaBH4还原,环氧氯丙烷交联制备壳聚糖衍生物,用FTIR对改性壳聚糖进行表征,并用于2,4-二氯苯酚的吸附研究。测试了改性产物的溶解性能,考察了吸附时间、溶液pH值、酚的浓度以及改性剂用量等因素对氯酚吸附的影响。结果表明,水杨醛改性环氧氯丙烷交联壳聚糖不溶于水、酸和碱;在pH=7、吸附时间为2 h时,对2,4-二氯酚的吸附量为128.2 mg/g。     

改性活性炭处理含酚废水的研究

分别采用高温、超声波、化学试剂、掺杂壳聚糖方法对活性炭进行了改性处理,并将其用于含酚废水的处理。结果表明:四种改性处理方法的效果依次为掺杂壳聚糖改性〉化学试剂改性>高温改性〉超声波改性;当壳聚糖与活性炭的掺杂比例为1:6时,最大吸附量达到67.1 mg/g,与未改性前的最大吸附量相比提高了116.2%.     

负载壳聚糖的凹凸棒土对Ni~(2+)离子吸附性能的研究

天然凹凸棒土(AT)经盐酸和热处理制得活化凹凸棒土(MAT).以壳聚糖(CTS)改性MAT制得负载壳聚糖的凹凸棒土(CMAT)。用扫描电镜(SEM)和红外光谱(IR)对AT、MAT和CMAT的结构进行了表征。考察了Ni2+离子溶液的初始浓度、pH、吸附时间以及吸附温度对CMAT和CTS两种吸附剂吸附Ni2+离子性能的影响,得出了适宜的吸附条件。用IR对这两种吸附剂及其适宜条件下得到的吸附产物的结构进行了表征,并作了比较。结果表明,CMAT吸附Ni2+离子的适宜条件为:Ni2+离子溶液初始浓度40.0 mg/L,pH 4.03~6.04,吸附时间1.5 h,吸附温度25℃其最大吸附容量为15.2 mg/L;CTS吸附Ni2+离子的适宜条件为:Ni2+离子溶液初始浓度40.0 mg/L,pH3.00~4.12,吸附时间1.5 h,吸附温度35℃,其最大吸附容量为12.3 mg/L。在相同实验条件下,CMAT对Ni2+离子的吸附性能高于CTS,且二者的吸附行为均符合Langmuir吸附等温式。IR分析表明,CMAT对Ni2+离于的吸附包含化学吸附和物理吸附两个过程。而CTS主要是化学吸附。     

壳聚糖保鲜涂膜纳米SiO_x修饰工艺优化的研究

为了提高壳聚糖涂膜的力学性能和降低其透水率,实验先以十二烷基磺酸钠(SDS)活化纳米SiOx,随后将其加到壳聚糖乙酸溶液中,并用流延法制得分散比较均匀的壳聚糖纳米复合膜.此外采用二次回归旋转正交组合试验设计方法,优化了壳聚糖/纳米SiO(CTS/SiOx)复合涂膜的制备工艺条件,并通过红外(IR),X射线衍射(XRD)和电镜透射(TEM)手段对CTS/SiOx.复合涂膜的性能和结构进行袭征.优化结果表明,当壳聚糖(CTS)、活化纳米SiOx和单甘酯(Glyc)添加量分别为1.547 g、0.028 g和0.015 g时,CTS/SiOx,复合涂膜的性能达到最优,其拉伸强度和撕裂强度分别为54.85MPa和51.77kN·m-1,分别比未修饰壳聚糖单膜提高了63%和12%,而透水率却下降了73%.将制备的CTS/SiOx复合涂膜用于果蔬室温保鲜,保鲜质量和保鲜时间比对照组均有明显提高.     

Pseudospecific magnetic affinity beads for immunoglobulin‐G depletion from human serum

Magnetic poly(ethylene glycol dimethacrylate‐N‐methacryloyl‐(L )‐histidine methyl ester) [m‐poly(EGDMA‐MAH)] beads were prepared by suspension polymerization for the affinity depletion of immunoglobulin‐G (IgG) from human serum in a batch system. Elemental analysis of the magnetic beads for nitrogen was estimated as 70 μmol MAH/g polymer. IgG adsorption onto the m‐poly(EGDMA) was negligible. Higher adsorption value (up to 46.8 mg/g) was obtained in which the m‐poly(EGDMA‐MAH) beads were used. IgG adsorption capacity of the magnetic beads increased with an increase in the concentration of IgG. The maximum IgG adsorption was observed at pH 6.5 for MOPS buffer. IgG molecules could be repeatedly adsorbed and eluted with these adsorbents, without noticeable loss in their IgG adsorption capacity. Adsorption capacity decreased for both increasing salt concentration and temperature. In this study, we show that m‐poly(EGDMA‐MAH) beads (wherein IgG molecules bind directly with the matrix) can be used directly for affinity depletion without further modification. Higher adsorption value was obtained from human serum (up to 85.7 mg/g). The elution results demonstrated that the adsorption of IgG to the adsorbent was reversible. The depletion efficiencies for IgG were above 85% for all studied concentrations. Eluted portion was analyzed for testing the IgG removal efficiency by two dimensional gel electrophoresis. Eluted proteins include mainly IgG, and a small number of nonalbumin proteins such as apolipoprotein A1, serotransferrin, haptoglobulin, and α1‐antitrypsin. IgA was not identified in eluted fraction. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Probing Antigen-Antibody Interaction Using Fluorescence Coupled Capillary Electrophoresis

In this report, the use of fluorescence detection coupled capillary electrophoresis (CE-FL) allowed us to fully characterize the antigen-antibody interaction. CE-FL allowed separation of unbound quantum dots (QDs) and ligand bound QDs and also revealed an ordered assembly of biomolecules on QDs. Further, we observed FRET from QDs donor to DyLight acceptor, which were covalently conjugated with human IgG and goat anti-human IgG, respectively. The immunocomplex was formed and the mutual affinity of the antigen and antibody brought QDs and DyLight close enough to allow FRET to occur. This novel CE-based technique can be easily extended to other FRET systems based on QDs and may have potential application in the detection of antibodies.     

Concanavalin A immobilized magnetic poly(glycidyl methacrylate) beads for antibody purification

Concanavalin A (Con A) immobilized magnetic poly(glycidyl methacrylate) (mPGMA) beads in monosize and spherical for (1.62 μm in diameter) were used for the purification of human immunoglobulin G (IgG) from human plasma. Con A was immobilized by covalent binding onto the mPGMA beads. The maximum IgG adsorption on the mPGMA-Con A beads was observed at pH 6.0. The nonspecific IgG adsorption onto the plain mPGMA beads was very low (0.22 mg/g). Scatchard analysis of the adsorption isotherm for IgG on mPGMA-Con A beads showed an affinity constant (K_a) of 1.39 × 10⁵ M⁻¹ and a theoretical maximum adsorption capacity of 109.1 mg/g. An apparent IgG adsorption capacity of 66.2 mg/g was observed under the experimental conditions. IgG adsorption capacity from human plasma was observed as 48.0 mg/g. The adsorption of human serum albumin from plasma was 2.0 mg/g. The total protein adsorption was determined to be 50.0 mg/g. IgG molecules could be repeatedly adsorbed and eluted with the mPGMA-Con A beads without noticeable loss in the IgG adsorption capacity. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Antibody purification by concanavalin A affinity chromatography

Concanavalin A (Con A) immobilized poly(2‐hydroxyethyl methacrylate) (PHEMA) beads in a spherical form (100–150 μm in diameter) were used for the affinity chromatography purification of human immunoglobulin G (IgG) from aqueous solutions and human plasma. PHEMA adsorbents were prepared by suspension polymerization. Bioligand Con A was then immobilized by covalent binding onto PHEMA beads. The maximum IgG adsorption on the PHEMA/Con A beads was observed at pH 6.0. The nonspecific IgG adsorption onto the plain PHEMA adsorbents was very low (ca. 0.17 mg/g). Higher adsorption values (up to 54.3 mg/g) were obtained when the PHEMA/Con A beads were used from aqueous solutions. A higher adsorption capacity was observed for human plasma (up to 69.4 mg/g) with a purity of 82.5%. The adsorption capacities of other blood proteins were 2.0 mg/g for fibrinogen and 4.2 mg/g for albumin. The total protein adsorption was determined to be 76.0 mg/g. IgG molecules could be repeatedly adsorbed and desorbed with the PHEMA/Con A beads without noticeable loss in the IgG adsorption capacity. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 1202–1208, 2005     

b型流感嗜血杆菌多糖ELISA检测方法的建立

目的建立b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)多糖(Polyribosylribitolphosphate,PRP)的ELISA检测方法。方法用灭活的Hib菌免疫家兔,1周免疫4次,共免疫6周,采血,检测血清抗体滴度。对兔免疫血清进行非特异性抗体免疫吸附,纯化兔抗PRP抗体,用纯化的兔抗PRP抗体包被酶标板,HRP标记的羊抗兔IgG作为二抗,建立双抗体夹心ELISA法检测PRP。确定标准曲线的最佳线性范围,并对方法进行特异性、准确性和精密性验证。结果兔免疫血清的抗体效价可达1∶2 800。该方法的线性检测范围为0.030～0.500 ng/ml,检测限为0.030 ng/ml。该方法检测大肠杆菌上清蛋白、牛血清白蛋白、LB培养基及破伤风-乙脑结合疫苗均无特异性反应;检测0.500、0.250、0.150 ng/ml的PRP标准品试验内变异系数(CV)在1.16%～2.40%之间,回收率在93.2%～107.2%之间;试验间CV在1.57%～4.11%之间,回收率在101.3%～101.6%之间。结论该方法特异性、准确性和精密性均较好,可以特异性检测b型流感嗜血杆菌多糖。     

纤维结合蛋白的提纯、定量、活性检测及抗体制备

采用重复两次亲和层析法从血浆中提取纤维结合蛋白(简称FN)可达到电泳纯;免疫家兔,抗FN血清平均效价可达16~x;经吸收得单特异抗血清;再经(NH_4)_1SO_4沉淀、DE-AE-HW离子交换可制出高纯度抗FN IgG;建立了两种FN的定量测定方法:1.火箭电泳法,检查了89份健康人血浆,平均含量为293±45μg/ml;2.免疫浊度法,检测了20份血浆,平均值为344±52μg/ml。最后建立了促进BHK细胞伸展活性的检测法,敏感度可达5μg/ml,并初步应用于稳定性和热变性试验,为开展FN的应用提供了质控指标。对比了我们精制的FN与SIGMA试剂公司的FN,二者成分相同,活性也很相似。     

Electrospun poly(hydroxybutyrate)/chitosan blend fibrous scaffolds for cartilage tissue engineering

In this study, polyhydroxybutyrate (PHB) was blended with chitosan (CTS), and electrospun in order to produce more hydrophilic fibrous scaffolds with higher mass loss rates for cartilage tissue engineering application. First, the effects of diverse factors on the average and distribution of fiber's diameter of PHB scaffolds were systematically evaluated by experimental design. Then, PHB 9 wt % solutions were blended with various ratios of CTS (5%, 10%, 15%, and 20%) using trifluoroacetic acid as a co‐solvent, and electrospun. The addition of CTS could decrease both water droplet contact angle from ～74° to ～67° and tensile strength from, ～87 MPa to ～31 MPa. According to the results, the scaffolds containing 15% and 20% CTS were selected as optimized scaffolds for further investigations. Mass loss percentage of these scaffolds was directly proportional to the amount of CTS. Chondrocytes attached well to the surfaces of these scaffolds. The findings suggested that PHB/CTS blend fibrous scaffolds have tremendous potentials for further investigations for the intended application. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44171.     

H5N1亚型禽流感病毒免疫血清精制工艺的建立

目的建立马抗H5N1亚型禽流感病毒免疫血清的精制工艺。方法以硫酸铵盐析法提取免疫马血浆中的IgG抗体,以8～256μg/mgIgG胃蛋白酶(活性单位1:3000)进行消化,以阳离子交换层析柱纯化F(ab’)2抗体,并参照《中国药典》三部(2005版)要求,对试制的样品进行检定。结果经一步50%硫酸铵和多步33%硫酸铵盐析,获得了较为纯净的IgG抗体。8μg胃蛋白酶用量可完全消化1mgIgG抗体分子,阳离子交换方法分离F(ab’)2抗体纯度可达90%以上,高于常规工艺制备的抗体纯度。以此工艺试制的样品,各项质量指标均符合《中国药典》三部(2005版)质量标准。结论已初步建立了马抗H5N1亚型禽流感病毒免疫血清的精制工艺。     

抗戊型肝炎病毒IgG抗体定量线性标准品的标定及初步应用

目的建立抗戊型肝炎病毒(Anti-HEV)IgG抗体的定量线性标准品,并进行初步应用。方法利用抗-HEV IgG和抗-HEV IgM ELISA检测试剂筛选出1份抗-HEV IgG阳性血清L9,经基因1型和4型的HEV ORF2C-端抗原及239抗原进行Western blot确认后,用WHO定量标准品,由3个实验室协作标定,利用量反应平行线法计算其抗-HEV IgG的含量。考察已标定的L9血清的稳定性,并用所标定的1.5倍系列稀释的血清对国内外6家抗-HEV IgG试剂的灵敏度进行检测。选择一灵敏度较高的试剂,在其线性范围内取L9的5个稀释度作为抗-HEV IgG抗体定量线性标准,对高、中、低浓度的3份临床血清重复检测5次,考察其重复性;对实验感染猴的系列血清中抗-HEV IgG含量进行定量检测,考核该定量线性标准品的应用效果;并对每次定量试验中的线性方程进行分析,确定相关系数r值和斜率k值的范围。结果经国内外试剂检测筛选出的阳性血清L9与基因1型和4型的HEV ORF2 C-端抗原及239抗原均有阳性反应。经协作标定,L9血清抗-HEV IgG含量为16.9U/ml。L9血清在-20℃下保存6、12、18个月,2～8℃保存24、48、96h后,定量结果均在95%置信区间内,且抗-HEV IgG含量均未明显下降。6家抗-HEVIgG检测试剂灵敏度差异较大,范围为0.03～5.00U/ml。确定L9血清从0.42U/ml开始的5个1.5倍系列稀释度,作为某一试剂抗-HEVIgG抗体定量线性标准品。利用该线性定量标准检测高、中、低浓度的3份临床血清,定量结果重复性较好;对实验感染猴系列血清进行定量检测,结果可有效地反映抗体水平变化趋势;94%的定量检测试验,r≥0.98,1.15≥k≥0.95。结论已建立了抗-HEVIgG抗体定量线性标准品,可用于疫苗免疫原性评价和流行病学调查。     

Facile purification and characterization of recombinant human antithrombin III from transgenic goat milk

BACKGROUND: Antithrombin III (AT III) is a serine protease inhibitor that inhibits thrombin and the activated forms of factors X, VII, IX, XI and XII. Transgenic expression of therapeutic proteins in animal systems has gradually matured from laboratory scale to industrial practice, demanding efficient and scalable purification processes. The purification and characterization of recombinant human antithrombin III (rhAT III) from transgenic goat milk are described here. RESULTS: The rhAT III was purified by isoelectric precipitation, heparin affinity chromatography, and size exclusion chromatography, resulting in a 90.6% yield and > 99% purity. The goat β‐casein secretion peptide introduced to the rhAT III was cut off using enterokinase and removed by size exclusion chromatography using a Superdex 75 column. The primary structure, disulfide linkages, glycosylation sites, secondary structure and tertiary structure of the rhAT III were measured and found to be the same as those of the plasma‐derived AT III (phAT III). CONCLUSION: A facile process is introduced for the purification of rhAT III from transgenic goat milk. The rhAT III with high purity was achieved after an initial isoelectric precipitation step in which most of the bulk protein impurities are removed, followed by affinity chromatography and size exclusion chromatography. The rhAT III was demonstrated to have the same structure as phAT III. Copyright © 2011 Society of Chemical Industry