私たちは、彼女の優秀な技術支援のための純子和田に感謝しています。私たちは親切PVR - IgG2aのためのバキュロウイルス発現ベクターを提供するための教授昭夫野本に感謝しています。この研究は、補助金ポリオ撲滅の推進と、厚生労働省からの新興再興感染症に関する研究によって部分的にサポートされていました。

1。維持培地（MM）と増殖培地（GM）の調製 MMとGMの調製のために、1のポリオ実験室のマニュアルを参照してください。ウシ胎児血清（FCS）および10％FCS 2％添加したダルベッコ改変イーグル培地（DMEM）はそれぞれ、MMとGMの代替として使用することができる。 2。抗PVの抗体の調製<ol><li>抗PVの抗体（タイプ1、2、または3）の各バイアルに水（バイアルあたり0.5 mL）を追加することによって再構成する抗PVの抗体（RIVM PVタイピング抗血清）。別のラベルの付いたチューブでMMの4.5 mLにこの再構成された抗体溶液（0.5 mLをそれぞれ）を追加します。 注：抗PVの抗体の一部ロットが再構成されたフォーム（バイアルあたり0.5mLの）として提供されています。抗PVの抗体のような多くの場合は、再構成のための水の添加は必要ありません。 </li><li>再構成された抗- PVの抗体の等量を混ぜる。抗PV1 +2、2 +3、または3 +1の場合：各再構成された抗体のミックス1.8mlの（それぞれの混合物1.8 mLを+ 1.8 mLの= 3.6 mL）を。各再構成された抗体のミックス1.2 mlの（1.2 mLを+ 1.2 mLの+ 1.2 mLの= 3.6 mL）中：抗- PV1 +2 +3のため。 注：PVサンプルPVの異なる血清型の混合物を含む可能性。 PVの混合物と試料中のPVの、単一の血清型を識別するには、抗PVの抗体（すなわち1 +2、2 +3、3 +1、1 +2 +3）のプールが必要です。 </li><li>各抗PV（1 +2、2 +3、3 +1、または1 +2 +3）抗体（3.6 mLを各）（最後の18.2パーセントFCS）にFCS（0.72 mL）を1 / 5ボリュームを追加します。 注：FCSの添加は、抗体によって引き起こされるゼラチン粒子の非特異的凝集の減少のためです。 </li><li> -20℃でPAのアッセイで使用するまで抗体のプールを保持します。 </li></ol> 3。感作ゼラチン粒子の再構成<ol><li>感作ゼラチン粒子1バイアル（凍結乾燥粉末として提供される）にキットに含まれている再構成バッファーを1.5 mLを加え。 </li><li>穏やかなタッピングでよく混ぜる</li><li> 4で再構成された感作粒子℃を保つ再構成されたゼラチン粒子は、少なくとも2週間は4℃に保ち、しかしちょうどアッセイ前に準備する必要がアクティブである可能性があります。 </li></ol>重要な注意： 感作ゼラチン粒子の非凝集のイメージは粒子の多くによって異なる可能性があるため、感作ゼラチン粒子の異なるロットを混ぜて使用しないでください。各実験では、感作ゼラチン粒子の単一の多くを使用する必要があります。 4。 PV識別のためのPAアッセイ<ol><li>マイクロ滴定プレートを準備します。 1つのウイルスのサンプルを識別するために、我々は5ウェルを必要とする+少なくとも一つのネガティブコントロール（下のウイルスと抗体なし感作粒子、よく＃6。例ではウイルスと抗体のコントロールを参照しない）。井戸の総数は、アッセイで5 ×サンプル+少なくとも1つのコントロールです。 重要な注意： アッセイ内の少なくとも1つはウイルスと抗体の制御を取らないしてください。感作粒子の1バイアルは10ウイルスサンプルの識別のために約です。 </li><li>マイクロ滴定プレートに抗PV抗体またはGMの12μL/ウェルを追加。プレートの配置は、（また、下記の実施例を参照）、次のとおりです。抗体なしのサンプル（よく＃1と6）で、GMの代わりに抗PVの抗体12μLを加える。 重要な注意： 多くのPVのサンプルはこのアッセイで試験する必要がある場合は、マルチチャンネルピペットまたはディスペンサーには、このステップの参考になる。 <table border="1"><tbody><tr><td>よく第1位</td><td>よく第2位</td><td>よく第3位</td><td>よく、第1位</td><td>よく＃5 </td><td>よく＃6 </td></tr><tr><td> GM </td><td>抗P1 +2 +3 </td><td>抗P1 2 </td><td>抗P2 3 </td><td> ANI - P1 +3 </td><td> GM </td></tr></tbody></table></li><li> 12μL/ウェルのウイルスのサンプルを追加。ウイルスのない一つのサンプル（よく＃6）で、12 GMのμLの代わりにウイルスのソリューションを追加します。 注：ウイルスのサンプルは通常、感染した細胞、L20B細胞またはRD細胞の培養上清である。 重要な注意： 多くのPVのサンプルはこのアッセイで試験する必要がある場合は、マルチチャンネルピペットは、このステップの参考になる。 <table border="1"><tbody><tr><td>よく第1位</td><td>よく第2位</td><td>よく第3位</td><td>よく、第1位</td><td>よく＃5 </td><td>よく＃6 </td></tr><tr><td>ウイルスのサンプル</td><td>ウイルスのサンプル</td><td>ウイルスのサンプル</td><td>ウイルスのサンプル</td><td>ウイルスのサンプル</td><td> GM </td></tr></tbody></table></li><li>室温に4℃から再構成された感作粒子溶液を取り、優しく完全に粒子を一時停止する]をタップします。 重要な注意： 穏やかなタッピングによる粒子の完全な停止の後、粒子は懸濁粒子析出する前にプレートに直ちに追加する必要があります。TEとフォームの非均質な溶液。 </li><li>すべてのウェルに感作された粒子の25μL/ウェルを追加。 重要な注意： 感作粒子の添加後、プレートはすぐにサンプルの均一な反応を開始するために混合プレートに供されてください。従って、感作粒子の添加は、プレート内のサンプルの間でタイムラグを最小限に抑えるためにディスペンサーを使用して実行する必要があります。 </li><li>プレートミキサーの上にマイクロ滴定プレートを入れ、30秒間プレートを混ぜる。 </li><li> 2時間室温でテーブルの上に白い紙の上にマイクロ滴定プレート（目視観察の便宜のために）（15〜30 ° C）を入れて 重要な注意： 結果の観察と撮影の写真のための反応の間と後の板を移動しないでください。形成された凝集が安定していないと簡単にプレートを移動することによって乱さ。 </li><li>プレートの写真を撮る。 重要な注意： 写真の部分的なビューは、後で調べると解釈の結果のレビューのための十分なデータを与えることができなかった。いくつかの写真は、井戸の凝集像を中心にプレートの全ウェルをカバーするために注意が必要です。 </li></ol> 5。結果の解釈<ol><li>正と負の井戸の識別。ウイルスと抗体のコントロールなしのそれ（ゼラチン粒子の析出、非凝集）（図1および2）で試料中の凝集反応を比較する。はっきりしないウイルスと、抗体のコントロール（非凝集、ネガティブコントロール）と区別されている凝集のイメージはポジティブとして判断されるべきである、とネガティブコントロールに似ている凝集のイメージがネガティブと判断されるべきである。明確なだけ部分的な凝集反応を示すサンプルは、&quot;正（部分凝集）&quot;として肯定的な、しかしレコードとして判断することができます。 </li><li> PVの同定。 PVの血清型を識別するために、抗PV抗体を用いた正と負の井戸に基づいて結果を解釈する。結果の解釈の原理は、細胞培養系における中和試験（例えば、正の抗P1 2抗体のサンプルは、タイプ3のPVが含まれていることを示す）のそれと似ています。 </li></ol> 6。代表的な結果 PAアッセイPV識別の代表的な結果を図1に示されています。全てのPVの株は感作ゼラチン粒子（なし抗体サンプル）の凝集を形成する。対応する抗PVの抗体の存在下では、ウイルスおよびno抗体の制御なしに似てゼラチン粒子（非凝集）（ネガティブコントロール）の沈殿が観察された（例えば、PV1（セービン）抗P1 +2の存在下でまたは抗P3 1抗体）。 <img src="/files/ftp_upload/2824/2824fig1.jpg" alt="図1" /> 図1。 PV1（セービン）のPAアッセイによるPVの識別。ウイルスソリューション、PV2（セービン）、及びPV3（セービン）（1.2 × 10 8 CCID 12分の50μL、8.2 × 10 7 CCID 12分の50μLのウイルス力価、およびそれぞれ3.5 × 10 7 CCID 12分の50μLは、）PAのアッセイにより同定のために使用されていました。いいえウイルスとしない抗体のコントロールは、ゼラチン粒子（非凝集、ネガティブコントロール）の析出は認められなかった。 <img src="/files/ftp_upload/2824/2824fig2.jpg" alt="図2" /> 図2。正と負の井戸の解釈。PV1（セービン）のウイルスソリューション、PV2（セービン）、及びPV3（セービン）（2.4 × 10 8 CCID 25分の50μLのウイルス力価、1.7 × 10 8 CCID 25分の50μL、および7.3 × 10 7 CCID 25分の50μL、それぞれ）320分の1〜1 / 20希釈し、そしてその後、希釈した試料25μLを抗PV抗体なしでPAアッセイにより調べた。

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Polio Laboratory Manual (4th edition) WHO/IVB/04.10. , (2004).</li><li>Arita, M., Masujima, S., Wakita, T., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+particle+agglutination+method+with+soluble+virus+receptor+for+identification+of+poliovirus.">Development of a particle agglutination method with soluble virus receptor for identification of poliovirus.</a> J. Clin. Microbiol. 48, 2698-2702 (2010).</li><li>Bibb, J. A., Witherell, G., Bernhardt, G., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+cell+surface+binding+site.">Interaction of poliovirus with its cell surface binding site.</a> Virology. 201, 107-115 (1994).</li><li>Koike, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+poliovirus+receptor+protein+is+produced+both+as+membrane-bound+and+secreted+forms.">The poliovirus receptor protein is produced both as membrane-bound and secreted forms.</a> Embo J. 9. , 3217-3224 (1990).</li><li>Mendelsohn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+a+human+poliovirus+receptor+gene+into+mouse+cells.">Transformation of a human poliovirus receptor gene into mouse cells.</a> Proc. Natl. Acad. Sci. U S A. 83, 7845-7849 (1986).</li><li>Mendelsohn, C. L., Wimmer, E., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+receptor+for+poliovirus:+molecular+cloning,+nucleotide+sequence,+and+expression+of+a+new+member+of+the+immunoglobulin+superfamily.">Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily.</a> Cell. 56, 855-865 (1989).</li><li>Arita, M., Koike, S., Aoki, J., Horie, H., Nomoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+poliovirus+with+its+purified+receptor+and+conformational+alteration+in+the+virion.">Interaction of poliovirus with its purified receptor and conformational alteration in the virion.</a> J. Virol. 72, 3578-3586 (1998).</li><li>McDermott, B. M., Rux, A. H., Eisenberg, R. J., Cohen, G. H., Racaniello, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+binding+affinities+of+poliovirus+for+its+cellular+receptor.">Two distinct binding affinities of poliovirus for its cellular receptor.</a> J. Biol. Chem. 275, 23089-23096 (2000).</li><li>Pipkin, P. A., Wood, D. J., Racaniello, V. R., Minor, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+L+cells+expressing+the+human+poliovirus+receptor+for+the+specific+detection+of+polioviruses+in+vitro.">Characterisation of L cells expressing the human poliovirus receptor for the specific detection of polioviruses in vitro.</a> J. Virol. Methods. 41, 333-340 (1993).</li><li>Avoort, H. G. v. a. n. d. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+five+methods+for+intratypic+differentiation+of+polioviruses.">Comparative study of five methods for intratypic differentiation of polioviruses.</a> J. Clin. Microbiol. 33, 2562-2566 (1995).</li><li>Kilpatrick, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+group-,+serotype-,+and+vaccine+strain-specific+identification+of+poliovirus+isolates+by+real-time+reverse+transcription-PCR+using+degenerate+primers+and+probes+containing+deoxyinosine+residues.">Rapid group-, serotype-, and vaccine strain-specific identification of poliovirus isolates by real-time reverse transcription-PCR using degenerate primers and probes containing deoxyinosine residues.</a> J. Clin. Microbiol. 47, 1939-1941 (2009).</li></ol>

総司増島は、新製品設計部、富士レビオ株式会社の従業員です。

このPAアッセイのユニークな点はPV 2の検出のための抗PVの抗体の代わりに、PVRの分子の使用率です。これはPV 3-6のすべての3つの血清型に均一な親和性でPV感作ゼラチン粒子の特異的相互作用が可能になります。 PVRの単量体の形態は、ビリオン上の単一の結合部位（〜-8 M、10 -7の解離定数）7、にのみ中等度の親和性を有するので、私たちは、ゼラチン粒子の感?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>感、ゼラチン粒子</td><td>富士レビオ株式会社</td><td></td><td>テストキット</td></tr><tr><td>再構成バッファ</td><td>富士レビオ株式会社</td><td></td><td>テストキット</td></tr><tr><td>マイクロタイタープレート</td><td>富士レビオ株式会社</td><td></td><td>テストキット</td></tr><tr><td>抗PV抗体</td><td> RIVM </td><td></td><td></td></tr></tbody></table>

particle agglutination method for poliovirus identification

世界ポリオ撲滅イニシアティブでは、実験室診断は、急性弛緩性麻痺（AFP）症例の便検体からPVを分離し、識別することによって、重要な役割を果たしている。世界保健機関（WHO）に、世界ポリオ研究所ネットワーク、PV分離と同定は、現在、それぞれ細胞培養系およびリアルタイムRT - PCRを使用して実行されている。 PVのポスト撲滅の時代には、簡単かつ迅速な識別手順は、国立研究所でポリオ症例の迅速な確認のために有用であろう。本研究では、我々は、PVの識別のために開発した斬新なPAアッセイの手順が表示されます。このPAのアッセイは、PV受容体（PVR）分子とPVのすべての血清型に特異的かつ均一な親和性であるビリオンの相互作用を利用しています。手順は簡単です（反応プレート内の1つのステップの手順）と急速に（結果は反応の2時間以内に得ることができます）、および結果が視覚的に（ゼラチン粒子凝集反応の観察）観察される。

Watch this Scientific Journal Video about Particle Agglutination Method for Poliovirus Identification at JoVE.com

Particle Agglutination Method for Poliovirus Identification

ウイルスの受容体分子を利用して、最近開発された新たな粒子の凝集反応（PA）アッセイは、ポリオウイルス（PV）の迅速かつ容易に識別することができました。この記事では、PVの識別のためのPAのアッセイのための手順が表示されます。

ポリオウイルスの同定のための粒子凝集法

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

particle-agglutination-method-for-poliovirus-identification

Research

JoVE Journal

Immunology and Infection

15.2K ビュー.  National Institute of Infectious Diseases. ウイルスの受容体分子を利用して、最近開発された新たな粒子の凝集反応（PA）アッセイは、ポリオウイルス（PV）の迅速かつ容易に識別することができました。この記事では、PVの識別のためのPAのアッセイのための手順が表示されます。

ビデオ: Particle Agglutination Method for Poliovirus Identification

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

の識別のための1.5時間の手順腸球菌</em直接血液培養から>種

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

免疫・感染学

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

機能的モチーフとその結合パートナーのペプチドベース識別

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

タングウイルスに対する新規抗ウイルス剤の同定のためのアッセイ

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

非宿主抵抗性に関与する遺伝子の同定のためのVIGS媒介フォワード遺伝スクリーニング


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF質量分析法を使用した血液培養ブロスからのグラム陰性菌の迅速な同定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomoを用いた電子Cryotomography再建からのウイルスのエンベロープ糖タンパク質スパイクの平均化

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

の莢膜血清型<em&gt;肺炎球菌</em&gt;ラテックス凝集による

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

単離、同定、およびマウス胸腺上皮細胞の精製

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

抗ウイルス化合物の同定および評価のための早期のウイルス侵入アッセイ

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...