Chicken antibody against a restrictive epitope of prion protein distinguishes normal and abnormal prion proteins

Recently, we reported the application of a recombinant chicken IgY monoclonal antibody, Ab3-15, against mammalian prion protein (PrP), for the diagnosis of bovine spongiform encephalopathy in cattle. In this study, we have characterized a soluble, single-chain variable fragment (scFv) form of this antibody, sphAb3-15 using brain homogenates from mice. This sphAb3-15 antibody recognized denatured forms of both PrP(C) and PrP(Sc), and PrP(Sc) after PK-treatment, on Western blotting. In sandwich ELISAs, on dot blots and by immunoprecipitation, sphAb3-15 efficiently bound to PrP from normal brain homogenates, but weakly bound PrP from scrapie-infected brain homogenates. These results suggest that sphAb3-15 selectively recognizes PrP(C) under native conditions and that the epitope recognized by sphAb3-15 may undergo conformational changes during the conversion of PrP(C) into PrP(Sc).     

Challenges facing the development and use of protein chips to analyze the phosphoproteome

Recent advances in analytical methods, particularly in the area of protein microarrays, have brought the field of proteomics to the forefront of biological science. Protein arrays have shown to be useful for the multiplexed analysis of several hundreds of proteins in parallel. While much of the effort has focused on developing methods to identify expressed proteins, the identification of post-translational modifications is equally important for comprehensive proteome characterization. Protein phosphorylation constitutes a major type of post-translational modification that mobilizes a high number of genes, is involved in many crucial cell functions and largely contriubutes to the complexity of the proteome. One of the major challenges to analyze phosphoproteins using arrays is the availability of specific antibodies. Thus far, this has hampered the development of highly complex phosphoprotein arrays. This review discusses some of the recent progress made in the development of techniques and reagents to quantitatively determine sites of protein phosphorylation.     

单克隆抗体在植物研究中的应用

单克隆抗体是现代生命科学研究的重要工具。随着分子生物学的发展,单克隆抗体在植物研究中发挥着越来越重要的作用。本文综述了单克隆抗体在蛋白表达、蛋白定位、蛋白相互作用、植物成分的定性与定量、植物成分纯化、植物病害检测、标签抗体等方面研究中的应用。     

Generation of a rabbit V(H) domain antibody polyspecific to c-Met and adenoviral knob protein

Several types of bispecific antibodies with affinity to both adenoviral coat proteins and a targeted antigen have been developed with the aim of providing the specific delivery of adenoviral gene therapy vehicle. From a phage display library of combinatorial dAb2s (each with an anti-adenoviral knob protein V(H) fragment linked with an anti-c-Met V(H)), we serendipitously enriched and isolated a clone, JS5, that has polyspecificity such that it binds both the adenoviral knob protein and c-Met, despite having only one V(H) domain. Our indirect observations suggest that the polyspecificity of JS5 is developed through accumulation of antibody specificity. The method of sequential immunization of a rabbit, first with the adenoviral knob protein and then with target antigens, may provide a method by which monoclonal antibodies with stand-alone polyspecificity may be developed. Such targeted polyspecific antibodies could readily be used for re-directing adenoviral vectors to target cells.     

抗体与SARS研究

在非典型性肺炎（SARS）的研究过程中,抗体无论在临床医学领域,还是在基础病毒学研究领域都发挥着重要的作用。利用重组蛋白或者人工合成的多肽免疫得到的针对SARS病毒特异性的单克隆、多克隆抗体可以用于SARS病人的临床血清学检测、SARS的预防治疗,还可以用于病毒蛋白的功能性研究。因此,抗体在类似SARS这样的感染性疾病研究过程中有着极大的应用价值。     

生物制药的现状和未来(一):历史与现实市场

以基因工程产品、抗体工程产品和细胞工程产品为主要代表的生物制药产业是发展最快的高技术产业之一 ,最近 6年全球年销售额连续每年以 1 5 %～ 33%的速度增长 ,并在 2 0 0 4年突破 40 0亿美元。生物技术药物在治疗肾性贫血、白细胞减少、癌症、器官移植排斥、类风湿关节炎、糖尿病、矮小症、心肌梗死、乙肝、丙肝、多发性硬皮病、不孕症、粘多糖病、戈谢病、法布莱氏病、囊性纤维化、血友病、银屑病和脓毒症等发挥了重要乃至关键作用。综述生物制药的历史、现状和市场。     

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.     

Weak protein interactions and pH- and temperature-dependent aggregation of human Fc1

The Fc (fragment crystallizable) is a common structural region in immunoglobulin gamma (IgG) proteins, IgG-based multi-specific platforms, and Fc-fusion platform technologies. Changes in conformational stability, protein-protein interactions, and aggregation of NS0-produced human Fc1 were quantified experimentally as a function of pH (4 to 6) and temperature (30 to 77°C), using a combination of differential scanning calorimetry, laser light scattering, size-exclusion chromatography, and capillary electrophoresis. The Fc1 was O-glycosylated at position 3 (threonine), and confirmed to correspond to the intact IgG1 by comparison with Fc1 produced by cleavage of the parent IgG1. Changing the pH caused large effects for thermal unfolding transitions, but it caused surprisingly smaller effects for electrostatic protein-protein interactions. The aggregation behavior was qualitatively similar across different solution conditions, with soluble dimers and larger oligomers formed in most cases. Aggregation rates spanned approximately 5 orders of magnitude and could be divided into 2 regimes: (i) Arrhenius, unfolding-limited aggregation at temperatures near or above the midpoint-unfolding temperature of the C_H2 domain; (ii) a non-Arrhenius regime at lower temperatures, presumably as a result of the temperature dependence of the unfolding enthalpy for the C_H2 domain. The non-Arrhenius regime was most pronounced for lower temperatures. Together with the weak protein-protein repulsions, these highlight challenges that are expected for maintaining long-term stability of biotechnology products that are based on human Fc constructs.     

An automated immunoassay for early specificity profiling of antibodies

Antibody-based therapeutics are of great value for the treatment of human diseases. In addition to functional activity, affinity or physico-chemical properties, antibody specificity is considered to be one of the most crucial attributes for safety and efficacy. Consequently, appropriate studies are required before entering clinical trials.

High content protein arrays are widely applied to assess antibody specificity, but this commercial solution can only be applied to final therapeutic antibody candidates because such arrays are expensive and their throughput is limited. A flexible, high-throughput and economical assay that allows specificity testing of IgG or Fab molecules during early discovery is described here. The 384-well microtiter plate assay contains a comprehensive panel of 32 test proteins and uses electrochemiluminescence as readout.

The Protein Panel Profiling (3P) was used to analyze marketed therapeutic antibodies that all showed highly specific binding profiles. Subsequently, 3P was applied to antibody candidates from early discovery and the results compared well with those obtained with a commercially available high content protein chip. Our results suggest that 3P can be applied as an additional filter for lead selection, allowing the identification of favorable antibody candidates in early discovery and thereby increasing the speed and possibility of success in drug development.     

A monoclonal antibody reactive with an activated ras protein expressing valine at position 12

Activated ras transforming genes have been described in a variety of neoplasms and encode 21,000-Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown to contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.     

gcm蛋白的表达、纯化及多克隆抗体的制备

gcm(glial cells missing)是调控神经元细胞和神经胶质细胞相互转化的一个基因开关.在gcm功能缺损的突变体中,预期的神经胶质细胞发育成神经元细胞;而在gcm过表达的突变体中,预期的神经元细胞转化为神经胶质细胞.此外,gcm还调控血浆细胞发育.为了进一步研究gcm在发育中的功能,需要获得gcm蛋白并制备其抗体.根据已报道的gcm基因序列,以果蝇cDNA文库为模板进行PCR扩增得到gcm部分编码区序列,然后将其连接到pET-28a载体以获得原核表达载体.重组载体经酶切测序鉴定确认后,转化大肠杆菌(E.coli)BL21,并用IPTG诱导融合蛋白表达.采用Ni-IDA凝胶柱亲和纯化蛋白,将纯化的His-gcm融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体效价.获得的gcm原核表达重组融合蛋白及高效价的特异性兔抗gcm多克隆抗体,为gcm功能的进一步研究奠定了基础.     

人DFF45蛋白多克隆抗体制备及其初步应用

目的：表达、纯化带GST标签的DNA裂解因子45（DNA fragmentation factor 45 ,DFF45）融合蛋白并制备多克隆抗体。方法：构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activated Sepharose? 4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果：成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1:20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论：DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。     

Generation of antibodies against membrane proteins

The monoclonal antibody has become an important therapeutic in the treatment of both hematological malignancies and solid tumors. The recent success of antibody-drug conjugates (ADCs) has broadened the extent of the potential target molecules in cancer immunotherapy. As a result, even molecules of low abundance have become targets for cytotoxic reagents.     

Quantitative immunoblot assay for assessment of liposomal antibody conjugation efficiency.

Routine direct assessment of immunoglobulin (Ig)-liposome(lp) conjugation efficiency has been impeded by phospholipid interference with standard protein and immunoassay methods. Rabbit IgG conjugated to anionic liposomes was quantitated in immunoblots using computer image analysis techniques. Lp-coupled Ig was separated from free Ig by dialysis in disposable Spectra/Por units (MWCO 300 kDa). Differential Lowry protein assay (DLA) of the thiolated Ig reactant and the dialyzate provided an estimate of conjugation efficiency that was compared to the results of the immunoblot assay (IBA). The color response of Ig-lp in the IBA was about an order of magnitude greater than rabbit IgG alone, requiring the synthesis of an Ig-lp standard in which the Ig conjugation efficiency was assessed by radiotracer methodology. The use of the same standard in three colorimetric protein assays verified the accuracy of the IBA and demonstrated that the colorimetric assays could be employed to determine Ig-lp conjugation efficiency. In terms of sensitivity and specificity, however, the IBA is better suited for routine assessment of laboratory-scale Ig-lp conjugation efficiencies. The DLA was found to be an unsatisfactory measure of conjugation efficiencies because an interfering substance was apparently released by Ig-lp preparations.     

Nodal蛋白的表达、纯化及多克隆抗体制备

斑马鱼心脏发育模型中Nodal编码转录因子调节心脏的左右不对称发育,为了进一步研究Nodal信号途径在心脏发育中的调控作用和心脏疾病发生的分子机制,需要获得斑马鱼Nodal蛋白并制备其抗体.采用从斑马鱼心脏组织中提取RNA,通过反转录得到心脏组织各种表达基因的cDNA为模板,PCR扩增得到Nodal部分编码区序列,然后将其连接到pET-28a载体上获得原核表达.经酶切及测序鉴定后,转化Rosseta细菌,并用IPTG诱导表达融合蛋白,Ni-IDA凝胶柱亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blotting检测抗体.获得了Nodal原核表达重组融合蛋白及高效价的特异性兔抗Nodal多克隆抗体,为Nodal功能的进一步研究奠定了基础.     

Performance optimization of continuous countercurrent tangential chromatography for antibody capture

Recent studies have demonstrated that continuous countercurrent tangential chromatography (CCTC) can effectively purify monoclonal antibodies from clarified cell culture fluid. CCTC has the potential to overcome many of the limitations of conventional packed bed protein A chromatography. This paper explores the optimization of CCTC in terms of product yield, impurity removal, overall productivity, and buffer usage. Modeling was based on data from bench‐scale process development and CCTC experiments for protein A capture of two clarified Chinese Hamster Ovary cell culture feedstocks containing monoclonal antibodies provided by industrial partners. The impact of resin binding capacity and kinetics, as well as staging strategy and buffer recycling, was assessed. It was found that optimal staging in the binding step provides better yield and increases overall system productivity by 8–16%. Utilization of higher number of stages in the wash and elution steps can lead to significant decreases in buffer usage (～40% reduction) as well as increased removal of impurities (～2 log greater removal). Further reductions in buffer usage can be obtained by recycling of buffer in the wash and regeneration steps (～35%). Preliminary results with smaller particle size resins show that the productivity of the CCTC system can be increased by 2.5‐fold up to 190 g of mAb/L of resin/hr due to the reduction in mass transfer limitations in the binding step. These results provide a solid framework for designing and optimizing CCTC technology for capture applications. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:430–439, 2016     

发菜NdhK的抗体制备与蛋白表达

发菜是一种陆生蓝藻,分布于一些干旱和半干旱区域。其NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO₂吸收、围绕光系统Ⅰ的循环电子传递和细胞呼吸。为研究该物种中ndhK基因的功能,本研究利用特异性引物,通过PCR方法从发菜中扩增ndhK基因并克隆到原核表达载体pET-32a上,得到表达载体pET-32a-ndhK,将其转入大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,得到分子量大小为43 kDa的融合蛋白NdhK。随后,采用亲和层析,对融合蛋白进行纯化回收,并以此回收蛋白作为抗原进行免疫,制备NdhK的多克隆抗体。最后,利用Western blot蛋白免疫印迹对所得抗体的特异性进行验证。从而为进一步探索发菜ndhK基因的功能以及发菜中NDH-1复合体各亚基的作用进行前期准备。     

Cloning and expression of a prion protein (PrP) gene from Korean bovine (Bos taurus coreanae) and production of rabbit anti-bovine PrP antibody

A PrP gene, from a Korean bovine, exhibiting a nonsense and a missense polymorphism respectively at nucleotides 576 and 652 has been cloned. The latter resulted in Glu to Lys substitution at amino acid residue 218. After expression and purification of the recombinant bovine PrP (recBoPrP) with Glu218Lys substitution, a polyclonal antibody against this protein was raised. ELISA and Western blot analysis suggested that the recBoPrP obtained in this study had a unique conformation not presented in native PrP(C), and the polyclonal antibody recognized PrP in a conformation dependent manner. These reagents will be valuable tools for studying PrP conformation.     

基于单克隆抗体的肿瘤免疫疗法研究进展

一百多年前,"魔术子弹"学说首次提出了具有靶向特异性的抗体可以用来治疗疾病。此后,随着单克隆抗体制备技术的成熟,以及癌症血清疗法的发展,靶向肿瘤抗原的治疗性抗体开始进入临床,至今已有20余种抗体药物用于癌症的治疗。近两年,以免疫检查点蛋白拮抗剂、双特异性抗体、抗体药物偶联药物等为代表的新一代抗体药物,不断在治疗恶性肿瘤上取得突破性进展。本文回顾了抗肿瘤抗体的发展历程,总结了新一代抗体药物的作用机制与构建策略,以及主要临床副作用。并对基于抗体的肿瘤免疫疗法未来发展趋势进行了展望。     

蛋白质微技术及其在医学领域中的应用

蛋白质微阵列是生物芯片的一种,其主要优势在于应用平面上的有序排列的许多管、腔（孔）或各自独立的点来进行样本检测,使大量样本的平行分析成为可能。应用此技术可同时分析诸多蛋白质的生物化学活性、蛋白质与蛋白质间、蛋白质与DNA间、蛋白质与RNA间,以及蛋白质与配体间的相互作用,从而在临床诊断、药物研究、环境监测、食品卫生等方面显示出其广阔的应用前景。