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

我々は彼らの貴重な貢献のためのK.中垣、博士石井、博士鈴木、A.山形、K. Oofusaに非常に感謝しています。

1。 sGMRαの生産と精製<ol><li>ポリメラーゼ連鎖反応（PCR）により、ヒト胎盤cDNAライブラリーからsGMRαのcDNAを増幅する。 </li><li> Hisタグ別のPCRによるPCRアンプリコンの3&#39;末端に50塩基のバキュロウイルスポリヘドリン最後の5&#39; - UTR配列と27塩基のコーディングRGSを追加。 </li><li>プラスミドpMSG1.1MGに増幅されたPCR産物を挿入します。 </li><li>カイコPND - W1株の卵にpHA3PIGヘルパーのベクトルを?...

<ol>
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無細胞アッセイは、優れた再現性と速さとGM - CSF自己抗体の中和能を推定する。 GM - CSF自己抗体または患者の血清IgG分画による結合阻害は、このアッセイにより評価した。データは、無細胞アッセイの結合阻害とそれぞれTF - 1細胞​​を用いてバイオアッセイの増殖阻害との間の相関を示した。バイオアッセイは、広く利用され、しかし別の施設と我々はこの新しいシステムを使用すること?...

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

背景：これまで、我々はその中和能を示したが、GM - CSF自己抗体の濃度は、自己免疫性肺肺胞タンパク症（PAP）1-3患者の疾患の重症度と相関していたではない。肺におけるGM - CSFの生物活性の廃止は、4,5自己免疫PAPの可能性が高い原因であるとして、それはPAPと各患者の病気の重症度を評価するためのGM - CSF自己抗体の中和能を測定するために期待される。 今までは、GM - CSF自己抗体の中和能は、ヒト骨髄細胞またはGM - CSF 6月8日で刺激TF - 1細胞の増殖阻害を評価することによって評価されている。バイオアッセイ系では、しかしながら、一定の条件で細胞を維持するための技術的な問題に起因する別の研究室からのデータを、比較するだけでなく、信頼性の高いデータを得るためにしばしば問題になります。 目的：無細胞受容体結合アッセイを用いて細胞表面にGM - CSF受容体に結合するGM - CSFを模倣する。 方法：トランスジェニックカイコ技術は、高純度9月13日に組換え可溶性GM - CSF受容体α（sGMRα）のための大規模な量を得るために適用した。組換えsGMRαは、絹のタンパク質に融合されることなく、絹の糸の親水性のセリシン層に含まれていた、従って、我々は容易に中性の水溶液14,15との良好な純度で繭から抽出することができます。幸いなことに、GM - CSFとの結合に不可欠なオリゴ糖の構造は、他の昆虫や酵母によって生成されるものよりも人間のsGMRαの構造に、より類似しています。 結果：無細胞アッセイ系sGMRαを使用しては高い可塑性と信頼性でデータが得られた。 sGMRαにGM - CSF結合を用量依存sGMRαを使って、新しい無細胞アッセイ系は、中和活性の測定のためのより有用であることを示す、TF - 1細胞​​を用いたバイオアッセイと同様の方法でポリクローナルGM - CSF自己抗体によって阻害されたTF - 1細胞​​またはヒト骨髄細胞を用いたバイオアッセイ系よりGM - CSF自己抗体の。 結論：我々はGM - CSF自己抗体の中和能を定量化する無細胞アッセイを確立した。

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

我々は、受容体への顆粒球マクロファージコロニー刺激因子（GM - CSF）の結合を推定するために、無細胞受容体結合アッセイをデザインしました。それは、優れた再現性とGM - CSF自己抗体による可溶性GM - CSF受容体αに結合するビオチン化GM - CSFの競合的阻害を評価することを可能にします。

組換え可溶性GM - CSF受容体を用いてGM - CSF抗体の中和能を推定する無細胞アッセイシステム

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

バキュロウイルス発現系を用いた新規H7N9インフルエンザウイルス由来の機能組換えヘマグルチニンとノイラミニダーゼタンパク質の発現

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

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

ファージディスプレイされた合成抗体ライブラリーから、スケーラブルな高スループットの選択

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

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

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

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

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

マウスリンパ球標識による<sup&gt; 64</sup以下のためのターゲット設定&gt;のCu-抗体の受容体<em&gt;インビボ</em&gt; PET / CTによる細胞輸送

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

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

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

試金を使用して SIAT1 MDCK 細胞のインフルエンザ a (h3n2) ウイルス中和によるひと血清中の抗体を中和することを測定

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

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

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

体外分化マウス顆粒球マクロファージ ・ コロニー刺激因子 (GM-CSF)-(THGM) ヘルパー T 細胞の生産

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

クリプトコッカスの研究における顕微鏡技術と蛍光読解の補完的利用-アメーバ相互作用

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