三聚氰胺单克隆抗体不同纯化方法的比较

为了建立更适用于三聚氰胺单克隆抗体的纯化方法,分别采用了硫酸铵两次沉淀法、正辛酸-硫酸铵沉淀法、凝胶过滤层析法、亲和层析法、阴离子交换层析法对三聚氰胺单克隆抗体进行纯化。对纯化后的三聚氰胺单克隆抗体的纯度、浓度、效价进行鉴定,对比不同纯化方法之间的差异。结果证明亲和层析法对于三聚氰胺单克隆抗体纯化最为适宜。由于亲和层析法纯化纯度高,快速便捷,对三聚氰胺单克隆抗体损伤小,最为推荐。研究者也可结合自身条件,参考文中数据结果,根据实验需要自行选择适合自身的纯化方法。     

高压液相离子交换层析法测定糖化血红蛋白初步评价

目的：评价高压液相离子交换层析法测定糖化血红蛋白（GHbAlc）的临床应用。方法：采用高压液相离子交换层析法测定糖化血红蛋白。结果：参考范围在4．5％-6．2％。批间CV≤0．9％，批内CV≤0．7％。结论：高压液相离子交换层析法测定GHbAlc准确。精密度高，速度快，为医生调整糖尿病治疗方案提供确实可靠的依据。     

石杉碱甲多克隆抗体纯化方法的研究

目的：根据石杉碱甲多克隆抗体的性质,采用不同的方法对其进行纯化及性质鉴定。方法本实验主要采用硫酸铵沉淀法、辛酸-硫酸铵沉淀法和Protein A亲和层析法分别对石杉碱甲多克隆抗体进行纯化,并分别对纯化后的抗体纯度、蛋白量和效价进行检测。结果电泳结果示Protein A亲和层析法纯化后仅出现两条色带,无明显杂带,纯度高。饱和硫酸铵沉淀法、辛酸-硫酸铵沉淀法和Protein A亲和层析法纯化后的抗体蛋白含量分别为4.67mg· mL －1、6.89mg· mL－1和13.44mg· mL －1。 Protein A亲和层析法纯化后抗体的效价大于1∶256000,其性质未发生变化。结论相较于硫酸铵沉淀法、辛酸-硫酸铵沉淀法,Protein A亲和层析法纯化石杉碱甲多克隆抗体的纯度、蛋白含量、抗体效价更高,纯化效果更优。     

用亲和层析法分离纯化降纤酶

目的用亲和层析法从尖吻蝮蛇蛇毒中分离纯化降纤酶。方法用离子交换色谱、单克隆抗体亲和色谱技术进行分离纯化。结果得到电泳纯的降纤酶，其分子量为36000。结论用单克隆抗体亲和色谱法纯化降纤酶是切实可行的。     

免疫吸附亲和层析法分离纯化甲胎蛋白生活工艺研究

采用３～５个月胎龄的胚胎组织,通过盐析凝胶层析和亲和层析法分离纯化甲胎蛋白（ＡＦＰ）。结果表明,通过盐析沉淀血红蛋白和Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析脱盐,延长了亲和层析柱的使用寿命,亲和柱使用６次以上层析效果仍保持稳定。每个胚胎平无可分离得到ＡＦＰ０．７ｍｇ,回收率为４５．７１％。紫外吸收法测定抗体偶联率为９５．３８％。ＳＤＳ－ＰＡＧＥ显示ＡＦＰ在Ｍｒ６６０００处一条带十分明显,纯度较高。该方法     

利用纯化工艺去除降纤酶细菌内毒素

利用DEAE -Sepharose和亲和层析法 ,有效地去除降纤酶的细菌内毒素 ,生产出符合部颁标准的降纤酶     

胶体金免疫层析法和ELISA在HBsAg检测中的方法学评价

目的：对胶体金免疫层析法（GICA）和ELISA两种检测HBsAg方法进行方法学比较。方法：用国产HBsAg胶体金纸条和ELISA试剂同时检测血清标本，结果：与ELISA相比，胶体金免疫层析法检测HBsAg的灵敏度为96%，特异性为100%，符合率为97%，以上结果可以看出，胶体金免疫层析法与ELISA相比特异性基本一致，但胶体金免疫层析法灵敏度稍差，结论：国产HBsAg胶体金纸条与ELISA试剂相比有较高的符合率，但灵敏度有待于进一步提高。     

离子交换层析法去除血液制品中牛海绵状脑病病原体的研究

克-雅病是由牛海绵状脑病传播的脑致命性疾病。血友病病人通过浓缩Ⅷ因子治疗感染克-雅病的危险是存在的。本文概述了克一雅病的病因、分型、传播途径及血液制品的危险，介绍了苏格兰国家输血服务中心在提取高纯度Ⅷ因子之前用离子交换层析法去除克-雅病病原体的试验及结果。     

亲和层析法生产降纤酶

用自制的亲和层析柱纯人来自尖吻蝮蛇毒的降纤酶，可得到符合部颁质量标准的产品，降纤酶的ＳＤＳ－ＰＡＧＥ纯度为单一酶带，ＨＰＬＣ纯度为单峰，酶比活为２８８６Ｕ／ｍｇ，与离子交换层析和凝胶过滤法比较，亲和层析法更适合规模化生产降纤酶。     

高效液相色谱法与凝胶层析法检测人血白蛋白制剂中多聚体的…

对１２批人血白蛋白制剂分别以ＨＰＬＣ法重复测定５次及用凝胶层析法重复测定４次。结果表明：ＨＰＬＣ法（ＲＳＤ０．２３％－２．８６％）较凝胶层析法（ＲＳＤ１．４１％－５．７０％）结果准确；ＨＰＬＣ法样品不稀释，直接进样，每样仅需３０ｍｉｎ，操作简便易行，可作为白蛋白制剂中多聚体的含量测定方法。     

膝沟藻毒素Gonyautoxins的分离纯化膝沟藻毒素Gonyautoxins的分离纯化

目的从微小亚历山大藻(Alexandriun minutum Halim)Amtk 2中分离纯化膝沟藻毒素gonyautoxins。 方法 用凝胶色谱、离子交换色谱等方法从人工培养的微小亚历山大藻Amtk 2酸酒精萃取物中分离纯化膝沟藻毒素。结果从100 L Amtk 2培养液中获得(6.74±0.31)×109个藻细胞,其酸酒精萃取物经凝胶色谱分离首次在国内得到膝沟藻毒素GTX-4,GTX-1,GTX-3和GTX-2混合物(29.59±0.28) mg。从此混合物中取出4.06 mg经两次离子交换色谱纯化得到纯GTX-4(0.40±0.002) mg, GTX-1(5.95±0.03)×10^-2 mg,GTX-3(6.92±0.05)×10^-4 mg和GTX-2(0.11±0.005) mg。结论人工培养的微小亚历山大藻Amtk 2酸酒精萃取物通过凝胶色谱和两次离子交换色谱分离能够得到纯的膝沟藻毒素GTX-4,GTX-1,GTX-3和GTX-2。     

柱层析法提取门冬酰胺酶

目的　改良门冬酰胺酶提取工艺。方法　菌体经破壁处理后 ,经离子交换层析和亲和层析 ,将目的蛋白层析出来。结果　其效率远远高于传统的有机溶剂沉淀法。结论　操作方法简便 ,易于放大 ,是生产厂家提高效率、降低生产成本、提高竞争力的有效途径     

RAPID SEPARATION OF PROTEINS AND THEIR HIGHER-MOLECULAR FRAGMENTS BY MEANS OF SPHERON ION-EXCHANGES*

Ion-exchange derivatives are described, of a hydrophilic rigid macroporous glycolmethacrylate gel called Spheron, suitable for rapid high-performance liquid chromatography (HPLC) of proteins and their fragments. Their flow parameters are compared with those of ion exchange derivatives of cellulose and poly-dextran. The conditions for work with them are described (regeneration, cycling, equilibration, column packing) as well as the construction of a simple apparatus for medium-pressure ion exchange chromatography of proteins. The efficiency of these ion exchangers for the separation of proteins is illustrated with examples of chromatography of an artificial mixture of serum albumin, chymotrypsinogen and lysozyme. Chromatography of cyanogen bromide fragments of serum albumin and the A and B chains of oxidized insulin showed that the method can be applied in chromatography on higher molecular protein fragments. A review of all proteins, including technical enzymes, which have already been chromatographed on Spheron ion exchangers is also given. The prospects of Spheron ion exchangers for HPLC of proteins and their fragments are briefly discussed.     

蝎镇痛抗肿瘤缬精甘肽的两个氨基酸残基对镇痛活性的影响

目的研究蝎镇痛抗肿瘤缬精甘肽(analgesic-antitumor peptide,AGAP)中2个氨基酸残基对其镇痛活性的影响。方法利用已构建成功的2个突变体W38G和R58D,采用金属离子螯合亲和层析和阳离子交换层析方法对突变体蛋白进行纯化。采用小鼠醋酸扭体法测定两个突变体的镇痛活性。结果在大肠杆菌BL21(DE3)中实现了可溶性表达。采用金属离子螯合层析和阳离子交换层析方法获得了电泳纯样品。突变体W38G镇痛活性与未突变重组蛋白(recombinant analgesic-antitumor peptide from Buthus martensii Karschr,BmK AGAP)相同,突变体R58D镇痛活性降低。结论突变体与rBmK AGAP相比镇痛活性发生变化,说明所选取的突变位点对蝎镇痛抗肿瘤缬精甘肽的镇痛活性具有一定的调节作用。     

A newly-detected reductase from Rauvolfia closes a gap in the biosynthesis of the antiarrhythmic alkaloid ajmaline

A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.     

High and low anticoagulant activity heparins. Preparation in large scale and degree of complexation with antithrombin III

Heparin was separated into fractions with high and low anticoagulant activities by selective barium precipitation, gel filtration and ion exchange chromatography methods. Enrichment in the anticoagulant activities were observed in fractions with high molecular weight or high degree of sulfation. The combination of selective barium precipitation and ion exchange chromatography led to the preparation of heparins with very high anticoagulant activities (300 IU/mg). These heparins have a high affinity for antithrombin III (greater than 80%) and were undistinguishable from those prepared by antithrombin III affinity chromatography regarding molecular weight, sulfate/hexosamine ratio, degree of binding to antithrombin III, electrofocusing pattern and anticoagulant activity. These methods allow now the preparation in industrial scale of heparins with high anticoagulant activities for in vivo studies.     

猪肝超氧化物歧化酶的纯化及其部分性质研究

采用cu~(2+)存在下,以低浓度氯化钠与肝脏匀浆、两次70℃水浴加热、硫酸铵盐析、丙酮沉淀和DEAE-Cellulose离子交换层析分离等方法,从猪肝脏中纯化出超氧化物歧化酶.经鉴定和性质测定表明,该酶为Cu·Zu-SOD,分子量320000,热稳定温度为75℃,在pH5.0～9.0有较好的稳定性.     

高效液相色谱法在合成多肽分离与纯化中的应用

近年来，多肽药物化学形成并迅速发展成为药物化学的重要分支。而随着分离纯化技术的进展，又大大加快了新活性多肽的发现和合成速度。在多肽合成中．许多杂质显示与产物类似的性质，随着肽链的增长，分离的难度也增大，因此纯化的方法和工艺显得异常重要。本文综述了凝胶过滤色谱、离子交换色谱和反相色谱等高效液相色谱技术在合成多肽分离与纯化中的设计和应用。     

Separation of biological proteins by liquid chromatography

After the success of human genome project, proteome is a new emerging field of biochemistry as it provides the knowledge of enzymes (proteins) interactions with different body organs and medicines administrated into human body. Therefore, the study of proteomics is very important for the development of new and effective drugs to control many lethal diseases. In proteomics study, analyses of proteome is essential and significant from the pathological point of views, i.e., in several serious diseases such as cancer, Alzheimer’s disease and aging, heart diseases and also for plant biology. The separation and identification of proteomics is a challenging job due to their complex structures and closely related physico-chemical behaviors. However, the recent advances in liquid chromatography make this job easy. Various kinds of liquid chromatography, along with different detectors and optimization strategies, have been discussed in this article. Besides, attempts have been made to include chirality concept in proteomics for understanding mechanism and medication of various disease controlled by different body proteins.Abbreviations: ACN, acetonitrile; AIEC, anion exchange chromatography; CEC, capillary electro-chromatography; CIEF, capillary isoelectric focusing; CSF, cerebrospinal fluid; 2D-nano LC, two-dimensional nano liquid chromatography quadrupole; Q-TOFMS/MS, time-of-flight tandem-mass spectrometry; EC, electro-chromatography; ESI-LC–MS, electrospray ionization liquid chromatography–mass spectrometry; FA, formic acid; FLP, FMRF amide-like peptide; GPI-APs, glycosylphosphadylinositol anchored proteins; GSH, glutathione stimulating hormone; GSTs, glutathione-S-transferase isoenzyme; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; ICAT, isotope coded affinity tag; IEF-SEC, isoelectrofocussing size-exclusion chromatography; IMCD, inner medullary collecting duct; LC–MS, liquid chromatography–mass spectrometry; LC-Q-TOF, liquid chromatography-quadrupole time-of-flight tandem mass; MS/MS, spectrometry; LC-dual ESI, liquid chromatography dual electrospray ionization-Fourier transform; FT-ICR-MS, ion cyclotron resonance-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization-time-of flight; MFGM, milk fat globule membranes; MMA, mass measurement accuracy; MPC, mesenchymal progenitor cell; NLFs, Nasal lavage fluids; NLP, neuropeptide like protein; PC2, prohormone convertase-2; PS II, photosystem II; RPLC, reversed phase liquid chromatography; SCX, strong cation exchange; SEC, size-exclusion chromatography; TFA, trifluoroacetic acid; TIC, total ion current; TRAF, tumor necrosis factor receptor