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

64.3K 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

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

細菌病原体脂肪酸を膜リン脂質に取り込む研究のための鶏卵黄からのリポタンパク質粒子の単離

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

生化学

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

鶏卵からの外側および内側のペリビテリンサブ層の機械的分離とタンパク質可溶化

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

カッパ（Vκ）に対する抗体の結合特異性軽鎖を含むヒト（IgM抗体）抗体：ポリシアル酸（PSA）ケーススタディとしてNCAMに添付

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

免疫・感染学

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

卵黄抗体に基づくサイトメトリービーズアッセイの標準化

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

ブロイラーチック胚からの前駆脂肪細胞の単離

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

発生生物学

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

生物工学

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

チャイニーズハムスター卵巣細胞における迅速な抗体糖工学

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

抗体生成:ハイブリドーマを用いたモノクローナル抗体の産生

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

高等生物学

免疫学

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www.jove.com/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

免疫沈殿ベースの技術:アガロースビーズを用いた内因性タンパク質の精製

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app.jove.com/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

チャイニーズハムスター卵巣細胞が産生するモノクローナル抗体を精製する技術

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...