赤霉素提取方法及得率比较研究

为了找出赤霉素液体发酵的提取最佳方法及其影响因素,对提取方法、溶剂、pH值、温度进行了系统的比较研究,得出以乙酸乙酯为溶剂,pH2.7,温度20℃的溶媒法的提取率相对较高,其最高得率为91.4%,与其它处理组比较,平均高出10%,工艺流程简单、技术易掌握,是符合生产要求的最佳工艺。     

山药糖蛋白提纯工艺

用正交试验优化了水浸提-醇沉淀法提取山药糖蛋白的工艺条件,通过DEAE-52离子交换层析柱和Sephadex G-75葡聚糖凝胶对粗提物进行纯化.实验结果表明,较优的提取条件是:提取温度65 ℃,时间2h,浸提次数3次,料水比1∶15.此条件下糖蛋白粗品提取率为1.23%,各因素的影响程度顺序是温度>料水比>时间>浸提次数.粗提物经DEAE-52,SephadexG-75柱层析纯化得到两个糖蛋白组分.     

脱脂对不同生长期苜蓿膳食纤维含量及其提取得率的影响

目的：研究脱脂对苜蓿膳食纤维含量、构成及其提取得率的影响,为该方法在苜蓿膳食纤维提取中的合理应用提供科学依据。方法：以初花期和结荚期苜蓿草粉为原料,研究脱脂前后苜蓿草粉中膳食纤维含量、构成及其提取得率的变化规律。结果：苜蓿膳食纤维以水不溶性膳食纤维（IDF）为主,开花后苜蓿IDF含量占膳食纤维总量（TDF）的90%以上;脱脂后的初花期和结荚期苜蓿草粉中,IDF含量分别达72.88%、73.62%,比脱脂前分别提高61.38%、38.12%;IDF在TDF中的比例分别由90.09%、92.82%提高至95.19%、95.71%,IDF提取得率分别达61.52%、72.11%,比脱脂前分别提高69.94%、48.62%;脱脂过程中水溶性膳食纤维（SDF）流失严重、含量降低,SDF提取得率下降92.68%和92.29%。结论：脱脂方法适用于结荚期苜蓿IDF提取原料的前处理,但不适于苜蓿SDF的提取。     

条斑紫菜R-藻红蛋白提纯工艺研究

对条斑紫菜叶状体R-藻红蛋白提纯方法进行了研究和分析。首先分别采用冻融法、化学试剂法、溶胀法等单一及组合细胞破碎方法对条斑紫菜细胞进行破碎,结果表明溶胀 组织捣碎法效果最佳。其次用25%～60%饱和度范围内的硫酸铵沉淀法粗提藻红蛋白,发现25%～45%硫酸铵分步沉淀法效果最好,再经结晶法盐析纯度(D565nm/D280nm)达到2.088。盐析液用SephadexG-25层析柱脱盐,再经羟基磷灰石(HA)柱层析,纯度可达到4.98。R-藻红蛋白的最大吸收峰在565nm,其室温荧光发射峰为578nm。SDS-PAGE结果显示,R-藻红蛋白可分为α、β两个亚基,Mr分别为17.0×103和19.0×103。     

一种提纯粗茶皂素的简易方法

对粗茶皂素的提取纯化工艺进行了研究。以质量分数为70％的粗茶皂素为原料,经2％NaOH溶解、盐酸酸析、95％乙醇溶解、丙酮沉析工艺得到精制茶皂素。检测结果表明,茶皂素质量分数大于95％,提取率大于80％,这种简便易行的工艺得到的茶皂素可作为开发新型植物灭螺剂的原料。     

一种高纯度、高得率的质粒DNA纯化法

一种高纯度、高得率的质粒ＤＮＡ纯化法汤乃梅,王晓民,韩济生（北京医科大学神经科学研究中心,北京１０００８３）关键词质粒ＤＮＡ纯化本文介绍一种方法,它不仅可以得到高纯度质粒ＤＮＡ,而且在同等纯度下的得率也高于其它方法,其操作流程如下：１．５００ｍｌ菌液...     

DM-130树脂对甘草酸的吸附性能及提纯应用研究

探讨了大孔吸附树脂DM-130对甘草酸的吸附性能及原液浓度、pH值、流速对此树脂吸附性能的影响.结果表明,DM-130树脂对甘草酸的吸附性能好,易于洗脱,分离效果好,产品纯度可达94.676%;正交试验表明,pH为5.4±,原液浓度为10 mg/ml,以2 BV/h的流速为最佳处理;洗脱液采用3 BV 10%的乙醇最经济.     

利用FPLC系统结合自装层析柱纯化兔血清IgG

为了建立一种简便、成本低的纯化兔血清IgG的实用方法,采用FPLC系统结合实验室常规手段填充的Sephacryl-S200凝胶柱和DEAE-Sephadex A-50离子交换柱进行兔血清IgG的纯化,结果表明,在流速为0．5ml／min时,此方法可以纯化得到高纯度的兔IgG;脱盐柱Sephadex G-25重复实验证明此实验方法具有很好的稳定性;并且与商品化的层析柱相比,成本大为下降;此方法主要的不足之处是层析柱的流速较低,为0．5ml／min左右。该方法可以推广到其他生物大分子的纯化中去。     

一种简便可较大量提取血清IgG的方法

用辛酸沉淀法结合应用少量阴离子交换剂纯化了四种动物抗血清及正常猪、羊血清的IgG。本法与硫酸铵法、亲和层析法纯化的IgG均呈现相同的电泳纯条带。本法简便、经济、回收率好,可较大量提取血清IgG。提纯过程保存抗体原有生物活性。     

Purification of IgG and albumin from human plasma by aqueous two phase system fractionation

The current shortages in human plasma products at global levels justify the development of new, cost effective plasma fractionation methods. We have developed a fractionation process to obtain immunoglobulin G (IgG) and albumin‐enriched fractions based on polymer‐salt aqueous two phase system (ATPS). A small‐scale (0.02 L) ATPS composed of polyethyleneglycol 3350 (PEG), potassium phosphate and sodium chloride, at pH 6.1, was evaluated and subjected to 50‐fold scale‐up (1 L). Further purification of the fractions was performed using caprylic acid precipitation and ion exchange chromatography. Similar yield and purity were obtained at both small and large scales. IgG precipitated in the PEG rich upper phase at 83% recovery and 2.75‐fold purification factor. An 81% pure albumin fraction was obtained in the salt rich bottom phase with a 91% yield. After polishing, IgG was obtained at a recovery of 70% and a purity of 92%. Corresponding values for albumin were 91% and 90%. This IgG and albumin fractionation technology deserves further evaluation as it may represent a potential alternative to conventional plasma fractionation methods. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1005–1011, 2012     

静注人免疫球蛋白中IgG含量检测方法的探讨

为了更好地进行生产质量控制,快速、准确的测定静注人免疫球蛋白中的IgG含量。实验中对2001~2008年的静注人免疫球蛋白中的IgG含量检测结果进行了抽样统计分析。分析结果表明:样品40倍稀释后测得的吸光值与标准曲线第三点的吸光值无显著性差异;样品40倍稀释后测得的IgG含量与样品进行20、30、40倍三个稀释度测得的IgG含量的平均值无显著性差异。从而证明,样品40倍稀释后测得的IgG含量可以代表该批样品的IgG含量,无需进行20、30倍的稀释。检测方法会更省时、省力、省材料。     

双峰驼血清IgG亚型的分离纯化及多克隆抗体的制备

双峰驼IgG亚型包含IgG1、IgG2和IgG3,其中IgG2和IgG3为重链抗体,在结构上与IgG1存在显著差异。为获取双峰驼血清中的IgG1、IgG2和IgG3,并分析其抗原特异性和抗体特异性,本文交替使用Protein A和Protein G亲和层析柱,对其分离纯化,并通过聚丙烯酰胺凝胶电泳进行鉴定;之后分别制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体,通过ELISA对制备的多克隆抗体的效价进行测定;最后应用Western blot评估这三个亚型多克隆抗体的特异性,进而对双峰驼血清中IgG1、IgG2和IgG3的抗原特异性进行分析。结果表明,应用Protein A和Protein G亲和层析柱成功分离纯化出双峰驼血清中的IgG1、IgG2和IgG3;并制备兔抗双峰驼IgG1、IgG2和IgG3的多克隆抗体效价均在1∶10000以上,并且所获得的多克隆抗体分别与IgG1、IgG2和IgG3之间均存在交叉反应,但兔抗双峰驼IgG1多克隆抗体较其它两个亚型多克隆抗体特异性低。结果证明,双峰驼IgG1、IgG2和IgG3均具有良好的免疫原性,三者结构虽存在显著差异,但其抗原特性类似。     

Rapid blood clearance of mouse IgG2a and human IgG1 in many nude and nu/+ mouse strains is due to low IgG2a serum concentrations

 We reported previously that the blood clearance of injected mouse IgG2a was extremely rapid in many strains of nude and nu/+ mice. In an attempt to determine the cause of this phenomenon, the levels of endogenous IgG2a in the blood of these mice was assayed. It was found that the serum level of IgG2a was extremely low in many of these mice, below 50 μg/ml, which is 20–100 times lower than the expected normal value. Great heterogeneity between individual mice was observed in their blood level of IgG2a, and there was an excellent correlation between low blood IgG2a levels and rapid clearance of injected IgG2a. Thus, the blood IgG2a levels are so low that a novel, previously undescribed effect occurs, namely the rapid clearance of small amounts of injected IgG2a. The clearance is due primarily to binding sites in the spleen and liver. The low level of endogenous IgG2a is not due to the lack of a thymus, since it occurs in nu/+ as well as nude mice, but can probably be attributed to the very clean environment in which these mice are raised. In assays of sera from approximately 50 mouse strains, low IgG2a levels were found in all nude colonies and also in some normal mouse strains. Some nude mice displayed relatively normal IgG2a clearance rates despite having low levels of endogenous IgG2a. In repeated bleedings of individual mice, IgG2a levels were found to fluctuate greatly. A similar clearance effect was observed with a human IgG1 Ab injected into mice. This rapid clearance of injected IgG, of certain subclasses, represents a practical problem for many experiments in which antibodies are used for diagnosis or therapy, and several methods of circumventing the problem are discussed. Received: 15 August 1977 / Accepted: 14 October 1997     

Synthesis and receptor binding of IgG1 peptides derived from the IgG Fc region

The IgG binding Fcgamma receptors (FcgammaRs) play a key role in defence against pathogens by linking humoral and cell-mediated immune responses. Impaired expression and/or function of FcgammaR may result in the development of pathological autoimmunity. Considering the functions of FcgammaRs, they are potential target molecules for drug design to aim at developing novel anti-inflammatory and immunomodulatory therapies. Previous data mostly obtained by X-ray analysis of ligand-receptor complexes indicate the profound role of the CH2 domain in binding to various FcgammaRs. Our aim was to localize linear segments, which are able to bind and also to modulate the function of the low affinity FcgammaRs, like FcgammaRIIb and FcgammaRIIIa. To this end a set of overlapping octapeptides was prepared corresponding to the 231-298 sequence of IgG1 CH2 domain and tested for binding to human recombinant soluble FcgammaRIIb. Based on these results, a second group of peptides was synthesized and their binding properties to recombinant soluble FcgammaRIIb, as well as to FcgammaRs expressed on the cell surface, was investigated. Here we report that peptide representing the Arg(255)-Ser(267) sequence of IgG1 is implicated in the binding to FcgammaRIIb. In addition we found that peptides corresponding to the Arg(255)-Ser(267), Lys(288)-Ser(298) or Pro(230)-Val(240) when presented in a multimeric form conjugated to branched chain polypeptide in uniformly oriented copies induced the release of TNFalpha, a pro-inflammatory cytokine from MonoMac monocyte cell line. These findings indicate that these conjugated peptides are able to cluster the activating FcgammaRs, and mediate FcgammaR dependent function. Peptide Arg(255)-Ser(267) can also be considered as a lead for further functional studies.     

Characterisation of IgG(T) serum antibody responses to two larval antigen complexes in horses naturally- or experimentally-infected with cyathostomins

Cyathostomins are the most common parasitic nematodes of horses. Larval stages, which inhabit the intestinal wall, are particularly pathogenic and can cause severe colitis and colic. Despite their clinical importance, diagnostic techniques for the prepatent stages do not exist. A method that could estimate mucosal infection intensity would have a major impact on the control and diagnosis of cyathostominosis. Here, serum IgG(T) responses to two larval antigen complexes of 25 and 20 kDa were quantified in horses with experimental infections, natural infections and in horses that presented with clinical larval cyathostominosis. In experimentally-infected animals, anti-25 kDa complex IgG(T) levels correlated positively with field exposure and with early third stage larval (r(s)=0.74, P=0.015) and total mucosal parasite (r(s)=0.78, P=0.010) burdens. In naturally exposed horses whose parasite burdens were quantified upon post-mortem examination, antigen-specific IgG(T) responses were significantly higher in infected than in uninfected horses (P=0.0001 and 0.002, for anti-25 and anti-20 kDa responses, respectively). In these animals, anti-25 kDa IgG(T) levels correlated positively with mucosal and lumenal burdens (P<0.05). IgG(T) responses to the 20 kDa antigen complex correlated positively with lumenal burdens (P=0.0043). In cases of larval cyathostominosis, antigen-specific IgG(T) levels were significantly higher than in uninfected ponies (P=0.002 and 0.0035, for anti-25 and anti-20 kDa responses, respectively). These results provide evidence that these two complexes contain antigens with potential as markers for prepatent cyathostomin infection.     

虎血清免疫球蛋白（IgG）的纯化及纯度、活性鉴定

目的 建立高纯度、高活性的虎血清IgG纯化方法。方法 用饱和硫酸铵沉淀虎血清得到IgG粗品;结合Hitrap Protein A亲和层析预装柱及阴离子交换层析法对粗品IgG进一步分离纯化,采用PAGE电泳和Western-Blot免疫印迹法鉴定IgG纯度和免疫活性。结果 80 mL虎血清亲和纯化得到84 mg IgG,阴离子交换层析纯化得到30 mg虎的IgG纯品。结论 建立了简便快速、纯度高、活性好的虎血清IgG的分离纯化方法,为虎血清IgG二级抗体的制备提供了高纯度、活性好的一级抗体免疫原。     

抗牛血清IgG单克隆抗体杂交瘤细胞株的建立及鉴定

用牛血清IgG免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,用含山羊血清的培养基培养细胞,上清用间接ELISA法筛选。获得4株能稳定分泌抗牛血清IgG的单克隆抗体杂交瘤细胞株,分别命名为1G5、2A8、3F5、4C5。其中2A8为IgG2a,其余3株为IgG1;腹水单抗的ELISA滴度均超过10-5;除3F5株单抗与山羊血清有交叉反应外,1G5、2A8、4C5株与人、马、猪、羊、兔、豚鼠等血清均不发生交叉反应;4株单抗与制备病毒性疫苗的基质液呈阴性反应;4株单抗识别分子量为160kD的牛血清IgG的两个不同抗原表位;4株单抗相对亲和力大小依次为4C5>2A8>1G5>3F5,相对敏感度依次为2A8>4C5>3F5>1G5;4株杂交瘤细胞株的染色体计数均大于90条,连续培养三个月以及冷冻保存半年后复苏,细胞生长良好。使用这些单抗建立的双抗体夹心法检测生物制品中的残留牛血清IgG。     

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.