この作品は、教育研究のドイツ連邦環境省（プロジェクトBiGRUDI [：R isikobewertung、U ltraschnelle D etektionウント私 dentifizierungフォンbioterroristisch relevanten Agenzien、13N9594のBi ologische G efahrenlagen]）からの助成金によって支えられて。我々は優秀な技術支援のためにB. Diemar（シャリテ- Universitätsmedizinベルリン、研究所こだわりPharmakologieを）感謝。

1。プロトコル - IGY抽出PEG -沈殿による図。 1 IGY -抽出手順の概略図を示します（表2参照）。プロトコルは、最初のポルソンらに記載されていた。 1980年。 すべてのステップは、ラテックスの手袋を使用して実行する必要があります。 <ol><li>卵殻は慎重に割れているし、卵黄はできるだけ白多くの卵として除去するために、&quot;卵黄のスプーン&quot;に転送されます。 </li><li>卵黄をろ紙に移し、残りの卵白を削除するにはロールバックされ、その後、卵黄の皮膚は、ランセットまたは類似の楽器（ピペットチップ）で切断されています。卵黄は、50 mlチューブに注入し、卵の体積は、（V1）に登録されます。 </li><li> PBSで2回の卵黄量が総量の卵黄（ΣV1+ V2）、その後3.5％PEG 6000（グラムでは、微粉炭）と混合されているが追加され、ボルテックス、10分のローリングミキサーで圧延が続いている。抽出手順のそのステップは2段階で、懸濁液を分離します。一つの相は、&quot;卵黄固体と脂肪性物質&quot;（ポルソンらのオリジナルの引用。1980）とIGYと他の蛋白質を含む水の相から構成されています。 </li><li>チューブは4℃で遠心分離している℃で20分（10,000 rpmで13000 × gで、ヘレウスMultifuge 3SR +、固定アングルローターに応じて）のためのC。上清（V3）は、折り畳まれたフィルターを通して注ぎ、新しいチューブに移している。 </li><li>グラム（新しいボリュームに応じて計算）で8.5％PEG 6000は、チューブに加え、ボルテックスし、手順3のようにローリングミキサーにロールバックされます。 </li><li>上清を廃棄していることの違いと手順4を繰り返します。 </li><li>ペレットは、慎重にガラス棒とボルテックスを用いて1mlのPBSに溶解させる。 PBSを10mlの最終容量（V4）に追加されます。溶液を12％のPEG 6000（w / vの、1.2グラム）と混合し、ステップ3（ボルテックス、ローリングミキサー）と同様に扱われます。 </li><li>手順6を繰り返しますし、800μlのPBS（ガラス棒とボルテックス）で慎重にペレットを溶解する。消えるのに空気泡を待って、透析のカプセルへの転送（ピペット）エキス。 400μlのPBSでチューブをリンスし、透析装置（V5）にボリュームを追加します。 （透析装置や膜の調製については付録を参照してください。） </li><li>抽出物を0.1％生理食塩水で一晩透析し（1,600 ml）をゆっくりとマグネチックスターラーにより撹拌する。翌朝は、生理食塩水をPBSで置き換え別の3時間透析されています。 </li><li>その後IGY -エキスをピペットで透析のカプセルから取り出され、2ミリリットルチューブに転送されます。最終容量は2ml（V6）程度である。 </li><li>サンプルのタンパク質含有量（mg / mL）を280 nmの（PBSで希釈1:50）で光学的に測定し、IGYのための1.33の吸光係数とランベルトベールの法則（図2参照）、図に基づいて計算されます。 。 4は、準備の品質（純度と回収率は約80％である）を示しています。 </li><li>それは、° C（​​-70 ° Cでサンプルを凍結していない）-20アリコートでサンプルを保存することをお勧めします。 </li><li> Joveのプロトコルで説明されているように最終的な準備の質は、単純なSDS - PAGEによって分析される<a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2。透析バッグを使用する前に、付録は、製造業者の推奨に従って、次の方法で準備する必要があります。 <ol><li> 20透析バッグは、30cmの片に切断し、2000mlのガラスビーカーに記載されています。 </li><li> 5mMのEDTA -ソリューションの1750ミリリットルが追加されます。 </li><li>ガラス漏斗は、袋をEDTA溶液でカバーされることを保証するために袋の上に配置されます。溶液を5分間（ホットプレート）加熱し、沸騰させる。ソリューションをデカントし、バッグを蒸留水で3回洗浄する。 </li><li>再び1750ミリリットルEDTA -溶液を加え、5分間煮沸し、上記のように蒸留水で3回洗浄する。 </li><li>最終的に透析バッグを蒸留水で10分間煮沸し、4℃で保存されています滅菌ピンセットを用いて透析バッグを取り出してください。 </li></ol> 3。代表的な結果<img src="/files/ftp_upload/3084/3084fig1.jpg" alt="図1" /> 図1。ポルソンらのオリジナルの記述に従ってポリエチレングリコール沈殿法によるIGY抽出の模式図。 1980年。 <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">拡大画像を表示するにはここをクリックしてください。</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="図2" /> 図2年齢依存性で免疫鶏の卵黄の合計- IgYの開発。合計IGY /卵とSD（n = 4の産卵鶏）の週平均がされ与えられた。 [ポーリーら 2009]から）。 <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="図3" /> 図3。拉乙別の抗原で免疫four鶏のngの容量の監視。矢印は、予防接種の日付を（[ポーリーら 2009]から）を示している。 <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">こちらをクリックして拡大画像を表示するには。</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="図4" /> 還元条件下での異なる卵から個々のIGYの準備の図4。ポリアクリルアミド- Gelelectrophoresis レーン1 - 分子量マーカー、レーン2 - IGY - 標準、レーン3-5 - プロトコルに記載したように調製した異なるIGYサンプル。これらはプロトコルの手順10によれば、最終的な製剤である。 HC - 重鎖、LC - 光chaines、？ - 35 kDaの（おそらくビテロゲニンIIの前駆体のC末端フラグメント、[。Klimentzou ら 、2006]）程度の分子量に対応するマイナー不純物。 <table border="1"><tbody><tr><td>&nbsp;</td><td> チキン21（抗原A） </td><td> チキン22（抗原A） </td><td> チキン19（抗原B） </td><td> チキン23（抗原C） </td></tr><tr><td>卵の数（合計IGY）1、2年間の期間</td><td> 625（38グラム） </td><td> 626（38グラム） </td><td> 545（33グラム） </td><td> 608（37グラム） </td></tr><tr><td>卵の数（合計IGY）、初年度</td><td> 345（20g）を</td><td> 304（16グラム） </td><td> 326（18グラム） </td><td> 308（17グラム） </td></tr><tr><td>卵の数（合計IGY）、二年目</td><td> 280（18グラム） </td><td> 322（21グラム） </td><td> 219（15グラム） </td><td> 300（20g）を</td></tr><tr><td> maxの％。可能な卵の数（初年度）2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> maxの％。可能な卵の数（2年） </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td>処理された卵の数</td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table>総IgYの1量は、図2のデータによると卵あたり60 mgのタンパク質の平均値によって卵の数を乗じて計算されます。 2以下 。年間可能な卵の数は、ブリーダーの情報によると325です。 表1。異なる抗原で免疫された鶏の産卵能力の2年間の統計。 （図も参照してください。3）二年の間に異なる抗原だけでなく、IGY -成果による免疫後4鶏の最大のパーセントで容量を敷設、[ポーリーら 2009]）。 <table border="1"><tbody><tr><td> 卵番号nr。 </td><td> ヘン数Nr。日付を敷設 </td><td> V1 [ML]卵黄 </td><td> V2 [ML] PBS </td><td> 1。 PEG PREC。 [G] </td><td> VOL [ML]の後に </td><td> 2。 PEG PREC。 [G] </td><td> ペレット </td><td> 3。 PEG PREC。 [G] </td><td> ペレット </td><td> VOL [ML] </td><td> 総タンパクmg / mlの </td><td> 補正した総タンパクmg / mlの </td></tr><tr><td></td><td></td><td></td><td> 2xV1ΣV1+ V2 </td><td> 3,5％[w / vの]ΣV1+ V2 </td><td> precは。、centrif。とろ過のV3 </td><td> 8,5％[w / vの] V3 </td><td> V4 mlのPBSに溶解</td><td> 12パーセント[W / V] V4 </td><td> mlのPBS V5に溶解</td><td> PBS V6に対して透析後、 </td><td> A280/ml </td><td> A280/ml：1.33 </td></tr><tr><td> 1 </td><td> H293 / 22.10。 </td><td> 15 </td><td> 30 45 </td><td> 1.58 </td><td> 31 </td><td> 2.63 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 35.0 </td><td> 26.3 </td></tr><tr><td> 2 </td><td> H293 / 23.10。 </td><td> 13 </td><td> 26 39 </td><td> 1.37 </td><td> 25 </td><td> 2.12 </td><td> 10 </td><td> 1.2 </td><td> 1.2 </td><td> 1.7 </td><td> 28.8 </td><td> 21.6 </td></tr></tbody></table> 表2。PEG沈殿の典型的なプロトコル

<ol>
	<li>Akita, E. M., Nakai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+purification+methods+for+the+production+of+immunoglobulins+from+eggs+laid+by+hens+immunized+with+an+enterotoxigenic+E.+coli+strain.">Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.</a> J. Immunol. Methods. 160, 207-214 (1993).</li><li>De Meulenaer, B., Huyghebaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+chicken+egg+yolk+immunoglobulins:+a+review.">Isolation and purification of chicken egg yolk immunoglobulins: a review.</a> Food Agricult. Immunol. 13, 275-288 (2001).</li><li>Klimentzou, P., Paravatou-Petsotas, M., Zikos, C. h., Beck, A., Skopeliti, M., Czarnecki, J., Tsitsilonis, O., Voelter, W., Livaniou, E., Evangelatos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+immunochemical+evaluation+of+antibodies+Y+for+the+poorly,+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly, immunogenic polypeptide prothymosin alpha.</a> Peptides. 27, 183-193 (2006).</li><li>Pauly, D., Dorner, M., Zhang, X., Hlinak, A., Dorner, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+laying+capacity,+immunoglobulin+Y+concentration,+and+antibody+titer+development+in+chickens+immunized+with+ricin+and+botulinum+toxins+over+a+two-year+period.">Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.</a> Poultry Science. 88, 281-290 (2009).</li><li>Polson, A., von Wechmar, M. B., van Regenmortel, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+viral+IgY+antibodies+from+yolks+of+immunized+hens.">Isolation of viral IgY antibodies from yolks of immunized hens.</a> Immunol. Commun. 9, 475-493 (1980).</li><li>Schade, R., Behn, I., Erhard, M., Hlinak, A., Staak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chicken egg yolk antibodies, production and application : IgY-Technology. , (2001).</li><li>Schade, R., Calzado, E. G., Sarmiento, R., Chacana, P. A., Porankiewicz-Asplund, J., Terzolo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+egg+yolk+antibodies+(IgY-technology):+a+review+of+progress+in+production+and+use+in+research+and+human+and+veterinary+medicine.">Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.</a> Altern. Lab Anim. 33, 129-154 (2005).</li><li>Witkowski, P. T., Bourquain, D. R., Hohn, O., Schade, R., Nitsche, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun-supported+DNA+immunisation+of+chicken+for+straightforward+production+of+poxvirus-specifiv+IgY+antibodies.">Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specifiv IgY antibodies.</a> J. Immunol. Methods. 341, 146-153 (2009).</li></ol>

動物実験はLandesamt毛皮お大事にウントSozialesベルリン（H 0069/03）によって定められたガイドラインおよび規制に準拠して行った。

適切なIGYの精製法の選択は、精製のスケール（実験室または工業用）、費用対効果、技術（実験装置）、および環境への影響（廃棄物管理）の影響を受けています。様々なIGY抽出法は、デMeulenaer＆Huyghebaert（2001年、レビューのためにも、シャーデらを参照してください。2005）によって詳細に検討された。一般的に、これらの方法は3つの主要なグループに分けることができます。 <ol><li> 降水方法：アンモニウムまたは硫酸ナトリウム、ポリエチレングリコール（PEG）、カプリル酸とcaragenean含む。 </li><li> クロマトグラフィー法：アフィニティークロマトグラフィー、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、thiophylic相互作用クロマトグラフィー、およびゲルろ過クロマトグラフィー。 </li><li> 限外ろ過 。 </li></ol> IGY準備の純度は​​方法の組み合わせによって増加することができます、例えば、PEG沈殿、アフィニティークロマトグラフィーと組み合わせることができます。いくつかの例では、最終的なアプリケーションに応じて、IgYの水抽出物は、良好な結果を（秋田＆中井1993）を達成するの​​に十分です。我々の経験によると、我々はPEG -沈殿により得るIGY -サンプルは、異なる免疫学的アッセイの多くで非常にうまくいった。 PEG -沈殿法を標準化することによって、一つの技術アシスタントは4と7 g当たり週の間に合計IgYの量に相当する週当たり約84卵を、処理することができます。簡単な計算：産卵鶏は、抗原で免疫される。いくつかのブースト予防接種後篇は30日の期間中1:50,000の力価と特異的抗体（AB）を生成します。 30卵X 2ミリリットルIGYエキスは、（プロトコルおよび図3を参照）3000リットルを意味する順番に3000リットルのAB -希釈（バケットの容量が10リットル）に相当する60ミリリットル合計IGY、、に対応抗体の溶液300mlバケットが満たされることができる。そう、このような巨大なAB -量のビューでは、一定の質と特異性の抗体プールを格納するためには問題ありません。この手法は、特に多特異性抗体で観察される電荷​​/ロットばらつきを低減します。 哺乳類の補体系のない活性化、HAMA（ヒト抗マウス抗体）、リウマチ因子やヒトの血液型抗原（heteroagglutininsの欠如）との交差反応性：量の側面にもかかわらず、IGY抗体は、哺乳動物のIgGと比較して、さらに利点があります。 IGY - ABの優れた利点は、いわゆる系統発生の距離です。系統学的距離は、同じ免疫場合でも、哺乳類とニワトリの抗体の特異性の間で頻繁に説明している違いがある理由です。哺乳類と鳥類の免疫系自体の違いに加えて、これら2つの動物クラスの系統発達の違いは、異なる抗体の特異性に寄与する。いくつかの著者は、鶏がしばしばより効率的にウサギが（審査のシャーデらA. 2005を参照）よりも系統発生的に高度に保存された哺乳類タンパク質またはペプチドに対してAbsを生成することが報告されている。結果として、保存された抗原はウサギ免疫システムに&quot;マスク&quot;のまま、そのためだけ弱いまたは&quot;サイレント&quot;応答を引き起こす可能性があります。さらに、非常に頻繁に、鶏やウサギが同じ哺乳類の抗原で免疫されている場合、鶏はほとんどウサギで達成することができるAB -特異性と反応する。抗体は、非侵襲的に卵黄から抽出されるので、また、動物福祉の側面が重要です。組み合わせで遺伝子銃（プラスミド）予防接種でIGY -生産（ウィトコフスキーら 2009）完全に非侵襲的です。 結論では、PEG -沈殿による鶏とIGY抽出における抗体の生産は非常に費用対効果が高く、最大100万分の1まで安定して力価を持つ非常に特異的な抗体の結果です。 鳥類と哺乳類との間の系統発生の距離が原因で、鶏肉は、例えばウサギとは対照的に高度に保存された哺乳類の抗原に対する特異的抗体を生成することができる。 IGY -技術によって得られた抗体の異常な量は、治療的/予防目的のために人間や動物用医薬品でIGY - Abを使用するためにも扉を開きます。

<ol><li> -14 kDの、カールロート社、カールスルーエ、ドイツを遮断バッグVisking、タイプ32分の27を、Dialyse </li><li>ファルコンチューブブルーマックス、50 mlのポリプロピレン製コニカルチューブ、ベクトンディッキンソン実験器具、フランクリンレイクスNY、アメリカ合衆国</li><li>フィルタMN 615は¼、∅150ミリメートル、カールロート社、カールスルーエ、ドイツヘレウスつ折り</li><li>マグネチックスターラー、Variomagモノ、H + P Labortechnik AG、オーバーシュライスハイム、ドイツ</li><li>マイクロDialyse -カプセル5,0 mlのカールロート社、カールスルーエ、ドイツ</li><li> Multifuge 3SR +、サーモサイエンティフィック、ランゲンゼルボルト、ドイツ</li><li>光度計UV - 160A、島津Deutschland GmbHは、デュースブルク、ドイツ</li><li>ポリエチレングリコール6000、Rotipuran薬局方。、カールロート社、カールスルーエ、ドイツ</li><li>予混合PBS -バッファ10倍、ロシュダイアグノスティックス社、マンハイム、ドイツ</li><li>ローラーミキサーSRT2、スチュアート、スタッフォードシャー、イギリス</li><li> Vibrofix VF2、ヤンケ＆クンケル、IKA - Labortechnik、シュタウフェンi.Br.、ドイツ</li><li>卵黄のスプーン、ファッケルマン（housholdの効果）、ドイツ</li></ol>

igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation

雌鶏は、IMワクチン接種（ 筋胸筋 、左と右、注入量0.5〜1.0ミリリットル）の手段によってまたは遺伝子銃プラスミド-予防接種による免疫することができる。抗原の免疫原性に依存し、高い抗体価は、（最大1:100,000 - 1:1,000,000）だけか3の後に達成することができます - 4ブースト予防接種を。通常は、鶏はその後産卵能力が低下し、約72週間連続して卵を産む。このプロトコルは、沈殿法の手段（PEG.ポルソンら 1980）で卵黄からの総IgYの抽出を説明します。方法は2つの重要なステップが含まれます。最初のものは、脂質の除去であり、2番目は第一段階の上清からの総IgYの降水量です。バッファ（通常はPBS）に対して透析した後IGY -エキスは一年以上は-20℃で保存することができます。抽出物の純度が80％程度であり、卵当たりの総IGYは、産卵鶏の年齢に依存し、40〜80 mgから変化する。最大80ミリグラム/卵（PEG沈殿に関する）に約40ミリグラム/卵から鶏の年齢と総IGYの含有量が増加する。年間鶏の産卵能力は325卵の周りです。 60mgの合計IGY /卵（表1参照）の平均IGYの内容に基づいて、20グラムの合計IGY /年間の総潜在的な収穫を意味する。

Watch this Scientific Journal Video about IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation at JoVE.com

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

このプロトコルは、ポリエチレングリコール沈殿によって、特に卵黄からの総IgYの抽出を説明し、IGY技術に関する一般的な情報を提供します。

IGY技術：ポリエチレングリコール（PEG）沈殿により卵黄からニワトリ抗体の抽出

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

igy-technology-extraction-chicken-antibodies-from-egg-yolk

Research

JoVE Journal

Immunology and Infection

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

64.1K ビュー.  Robert Koch-Institute. このプロトコルは、ポリエチレングリコール沈殿によって、特に卵黄からの総IgYの抽出を説明し、IGY技術に関する一般的な情報を提供します。

ビデオ: IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

の稀なケースでIgAおよび組織トランスグルタミナーゼ2抗体による表皮トランスグルタミナーゼの認識アカゲザル炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

免疫・感染学

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

からのDNA抽出のためのシンプルなChelexプロトコル<em&gt;ハマダラカ</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

ラテラルフロー技術を用いた血液学的悪性腫瘍患者における侵襲性肺アスペルギルス症の検出

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

ホット水 - フェノール抽出によるグラム陰性菌からの精製とリポ多糖の可視化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

鶏卵卵におけるインフルエンザウイルスの伝播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

ハイブリドーマ技術によってマウスモノクローナル抗体の作製

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

インフルエンザ血球凝集素抗体による Fc を介したエフェクター機能を評価する方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

細菌感染や解析のためのキイロショウジョウバエ幼虫血球の抽出

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...