Name: a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor
Uploaded: 2011-06-27T13:00:00
Description: Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

我们非常感谢他们的宝贵贡献，K. Nakagaki，博士阁下石井，铃木博士光，A.山形，光Oofusa。

1。生产和净化的sGMRα <ol><li>从人胎盘cDNA文库中扩增cDNA的聚合酶链反应（PCR）的sGMRα。 </li><li> 50基地5&#39; - UTR序列杆状病毒多角月底和27基编码的RGS His - tag的3&#39;末端的另一个PCR扩增PCR扩增。 </li><li>插入质粒pMSG1.1MG的扩增的PCR产物。 </li><li>注入鸡蛋蚕PND - W1株pHA3PIG助手载体的建设psGMR/M1.1MG。 </li><li>后部孵化G0期幼虫蛾25 ° C。 G1的屏幕获得通过交配，兄弟姐妹之间或与PND - W1在眼里MGFP表达，获...

<ol>
	<li>Arai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+neutralizing+capacity+of+GM-CSF+reflects+disease+severity+in+a+patient+with+pulmonary+alveolar+proteinosis+successfully+treated+with+inhaled+GM-CSF.">Serum neutralizing capacity of GM-CSF reflects disease severity in a patient with pulmonary alveolar proteinosis successfully treated with inhaled GM-CSF.</a> Respir Med. 98, 1227-1230 (2004).</li><li>Tazawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+and+lung+immunity+in+pulmonary+alveolar+proteinosis.">Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis.</a> Am J Respir Crit Care Med. 171, 1142-1149 (2005).</li><li>Inoue, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+large+cohort+of+patients+with+autoimmune+pulmonary+alveolar+proteinosis+in+Japan.">Characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in Japan.</a> Am J Respir Crit Care Med. 177, 752-762 (2008).</li><li>Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+autoantibodies+specifically+eliminate+granulocyte-macrophage+colony-stimulating+factor+activity+in+the+lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis.">High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis.</a> Blood. 103, 1089-1098 (2004).</li><li>Sakagami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+GM-CSF+autoantibodies+and+reproduction+of+pulmonary+alveolar+proteinosis.">Human GM-CSF autoantibodies and reproduction of pulmonary alveolar proteinosis.</a> N Engl J Med. 361, 2679-2681 (2009).</li><li>Raines, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+cloning+of+a+soluble+human+granulocyte-macrophage+colony-stimulating+factor+receptor.">Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.</a> Proc Natl Acad Sci U S A. 88, 8203-8207 (1991).</li><li>Williams, W., VonFeldt, J., Rosenbaum, H., Ugen, K., Weiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+soluble+form+of+the+granulocyte-macrophage+colony-stimulating+factor+receptor+alpha+chain+from+a+myelomonocytic+cell+line.+Expression,+biologic+activity,+and+preliminary+analysis+of+transcript+distribution.">Molecular cloning of a soluble form of the granulocyte-macrophage colony-stimulating factor receptor alpha chain from a myelomonocytic cell line. Expression, biologic activity, and preliminary analysis of transcript distribution.</a> Arthritis Rheum. 37, 1468-1478 (1994).</li><li>Prevost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(GM-CSF)+and+inflammatory+stimuli+up-regulate+secretion+of+the+soluble+GM-CSF+receptor+in+human+monocytes:+evidence+for+ectodomain+shedding+of+the+cell+surface+GM-CSF+receptor+alpha+subunit.">Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit.</a> J Immunol. 169, 5679-5688 (2002).</li><li>Iizuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+recombinant+mouse+monoclonal+antibody+in+transgenic+silkworm+cocoons.">Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.</a> FEBS J. 276, 5806-5820 (2009).</li><li>Iizuka, M., Tomita, M., Shimizu, K., Kikuchi, Y., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+enhancement+of+recombinant+protein+synthesis+in+transgenic+silkworms+by+a+5'-untranslated+region+of+polyhedrin+gene+of+Bombyx+mori+Nucleopolyhedrovirus.">Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.</a> J Biosci Bioeng. 105, 595-603 (2008).</li><li>Zou, W., Ueda, M., Yamanaka, H., Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+combinatorial+protein+library+displayed+on+yeast+cell+surface+using+DNA+random+priming+method.">Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method.</a> J Biosci Bioeng. 92, 393-396 (2001).</li><li>Tamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nat Biotechnol. 18, 81-84 (2000).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+silkworms+produce+recombinant+human+type+III+procollagen+in+cocoons.">Transgenic silkworms produce recombinant human type III procollagen in cocoons.</a> Nat Biotechnol. 21, 52-56 (2003).</li><li>Ogawa, S., Tomita, M., Shimizu, K., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon:+production+of+recombinant+human+serum+albumin.">Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.</a> J Biotechnol. 128, 531-544 (2007).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon.">A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.</a> Transgenic Res. 16, 449-465 (2007).</li><li>Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+alveolar+proteinosis+as+an+autoimmune+disease+with+neutralizing+antibody+against+granulocyte/macrophage+colony-stimulating+factor.">Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 190, 875-880 (1999).</li><li>Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis+express+a+factor+which+neutralizes+granulocyte-macrophage+colony+stimulating+factor.">Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor.</a> FEBS Lett. 442, 246-250 (1999).</li><li>Brown, C., Pihl, C., Murray, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+soluble+granulocyte-macrophage+colony-stimulating+factor+receptor:+identification+of+the+functional+ligand-binding+species.">Oligomerization of the soluble granulocyte-macrophage colony-stimulating factor receptor: identification of the functional ligand-binding species.</a> Cytokine. 9, 219-225 (1997).</li><li>Urano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+assay+to+estimate+the+neutralizing+capacity+of+granulocyte-macrophage+colony-stimulating+factor+autoantibodies.">A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.</a> J Immunol Methods. 360, 141-148 (2010).</li><li>Hayashida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+second+subunit+of+the+receptor+for+human+granulocyte-macrophage+colony-stimulating+factor+(GM-CSF):+reconstitution+of+a+high-affinity+GM-CSF+receptor.">Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.</a> Proc Natl Acad Sci U S A. 87, 9655-9659 (1990).</li><li>Hansen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+GM-CSF+receptor+complex+reveals+a+distinct+mode+of+cytokine+receptor+activation.">The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.</a> Cell. 134, 496-507 (2008).</li></ol>

无细胞检测估计具有优良的重现性和快速性的GM - CSF的自身抗体的中和能力。结合抑制GM - CSF的自身抗体或患者的血清IgG分数是评估这个实验。数据显示，结合抑制细胞免费检测和使用TF - 1细胞，分别为生物活性的生长抑制之间的关系。已被广泛使用的生物活性，但窝藏在比较数据之间不同的设施和不同的时间点，我们通过使用这个新的系统可避免的困难。 GM - CSF的结合sGMRα?...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

背景：在此之前，我们表明，中和能力，但没有GM - CSF的抗体浓度是在患者自身免疫性肺泡蛋白沉积症（PAP）的1-3疾病的严重程度相关。废止的GM - CSF在肺癌的生物活性可能导致自身免疫性PAP 的 4,5，它是有前途的GM - CSF自身抗体的中和能力的评估与人民行动党在每个病人的病情轻重程度来衡量。 到现在为止，已评定的GM - CSF自身抗体的中和能力评估人类骨髓细胞或TF - 1 细胞 6-8与GM - CSF的刺激的生长抑制。然而，在生物测定系统，它往往是问题，以获得可靠的数据，以及比较来自不同实验室的数据，由于技术上的困难，保持在一个恒定的条件下的细胞。 目的：为了模仿GM - CSF，GM - CSF受体细胞表面上的具有约束力的使用无细胞受体结合测定。 方法：转基因家蚕技术是获得高纯度9-13的重组可溶性GM - CSF受体α（sGMRα）为大量的应用。重组sGMRα载而不融合丝蛋白在亲水丝胶丝线层，因此，我们可以很容易地从良好的纯度蚕茧14,15与中性水溶液中提取。幸运的是，寡糖的结构，这是与GM - CSF结合的关键，更类似于人类sGMRα的结构比其他昆虫或酵母生产。 结果：无细胞检测系统使用sGMRα取得的数据，具有很高的可塑性和可靠性。 GM - CSF的剂量依赖性绑定sGMRα以类似的方式使用TF - 1细胞的生物活性，抑制多克隆GM - CSF的抗体，这表明我们的新的无细胞检测系统使用sGMRα是中和活性的测量更为有用GM - CSF的自身抗体比使用TF - 1细胞或骨髓细胞的生物测定系统。 结论：我们建立了无细胞的检测，量化的GM - CSF抗体的中和能力。

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

我们设计了一个无细胞受体结合实验，以估计约束力的粒细胞 - 巨噬细胞集落刺激因子（GM - CSF的）受体。它使我们能够评估竞争性抑制GM - CSF的抗体生物素标记的GM - CSF的可溶性GM - CSF受体α的结合，具有优良的重现性。

一个细胞检测系统估算的GM - CSF抗体的中和能力，采用重组可溶性GM - CSF受体

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

Research

JoVE Journal

Immunology and Infection

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

科研

免疫与感染

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

代和GM-CSF的鉴定源性肺泡状巨噬细胞和树突状细胞从小鼠骨髓

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

小鼠淋巴细胞通过标签<sup&gt; 64</sup&gt;的Cu-抗体受体靶向用于<em&gt;体内</em&gt;由PET / CT细胞运输

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

一种新型大鼠自然杀伤细胞无饲养系统的研究体外

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

体外小鼠粒细胞-巨噬细胞集落刺激因子 (gm-CSF) 的分化--生成 t 辅助器 (tHgm) 细胞

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...