私たちは、それぞれ、PA-824（pretomanid）、AZD5847、radezolidとtedizolid bedaquilineを提供するためのジョンソン・エンド・ジョンソン、TBアライアンス、アストラゼネカ、リブ-XとTrius治療に感謝したいです。ブレンダンプリドー、マシュー・ツィンマーマン、ステファン・ジャズウィン、エマ・レイ・フラド、ナンシールーエル、レヤン・リおよびダニエル・ウェイナー MALDI分析、生物分析方法、caseumサロゲートの調製、化学合成、およびウサギcaseumの単離と支持を提供しました。この作品は、センターオブエクセレンスのためにビル・アンド・メリンダ・ゲイツ財団とウェルカム・トラストからビル・アンド・メリンダ・ゲイツ財団、賞＃OPP1044966とOPP1024050からV・ダートイス、NIH共有計装グラントS10OD018072だけでなく、共同資金に資金調達を行いましたP・ワイヤットに発展途上国の疾患のためのリード最適化のために。

すべての動物研究は、NIAID（NIH）、Bethesda、MDの機関動物管理および使用委員会の承認を得て、国立衛生研究所の実験動物の管理および使用のガイドに従って実施した。 結核菌を含むすべての研究は、生物安全性封じ込めレベル3（BSL-3）の実験室で実施された。 1.ウサギ感染モデルとカゼムコレクション<ol><li> 以前に記載されたような鼻のみのエアロゾル曝露システムを使用して、 結核菌でニュージーランドの白色ウサギに感染させる。 12、13感染が12〜16週間進行するようにします。ウサギを35mg / kgのケタミンおよび5mg / kgのキシラジンで筋肉内に静置し、ウサギに0.22mL / kgのペントバルビタールナトリウムおよびフェニトインナトリウムを静脈内に安楽死させ、剖検を続ける。 </li><li>ピンセットとメスを使用して、胸部から肺を取り除く。vity。各肺葉から、個々の空洞および大きな壊死肉芽腫をメスを用いて切開する。空洞と肉芽腫の壁からカゼムを慎重にこすり落とす。使用準備が整うまで-20℃で2 mLスクリューキャップチューブに試料を計量し、記録し、保存する。 </li><li> 3メガラドのドライアイスで感染性のカゼウムサンプルをガンマ線照射して、BSL-2ラボでの使用に不快で安全なものにします。 </li></ol> 2.THP-1細胞からのCaseum Surrogateのインビトロ発生<ol><li> T175細胞培養フラスコ（80mL /フラスコ）中のRPMI1640培地（2mM L-グルタミンおよび10％ウシ胎仔血清）中のTHP-1単球を増殖させる。フラスコを5％CO 2雰囲気下、37℃で3〜4日間インキュベートする。 </li><li> T175フラスコから2本の50mLコニカルチューブで培養液を150 xgで5分間遠心します。上清を捨て、ペレットを10mLのRPMI 1640培地に懸濁する。 </li><li>この培養物5μLを45μLのtを含む1.5mLチューブにピペットライパンブルー。ピペッティングにより完全に混合する。 10μLを血球計算盤に移し、光学顕微鏡（倍率10倍）を用いて生存可能なTHP-1単球の数を数えた（染色していない）。培養液1mLあたりの生存細胞数を計算する。 RPMI培地で希釈して1.25 x 10 6 cells / mLの最終密度にします。 </li><li>大きな細胞培養プレート（50×10 6細胞/プレート）上に培養物40mLをロードする。 40μLの100μMPMA（エタノール中で調製したホルボール12-ミリステート13-アセテート）を加え、細胞をインキュベーター内で一晩付着させる。 注：PMAの最終濃度は100nMである。 </li><li>エタノール中の純粋なオレイン酸（OA）（0.89g / mL）を0.1Mの濃度（ すなわち、エタノール968.3μL中の31.7μLのOA）に希釈する。新鮮な予熱したRMPI培地でこの溶液を10 mMの濃度に希釈する。 37℃に予熱したRPMI培地中でこのOA懸濁液を0.4mM（最終使用濃度）に希釈する。 </li><li>既存のメディアと非メディアを削除する-adhered細胞を細胞培養プレートから、穏やかにはTHP-1マクロファージ（THP-M）を0.4 mMのOAの40ミリリットルを加えます。一晩インキュベーター中で37°Cでインキュベートします。 </li><li>視覚各THP-Mにおける多数の脂質体の介在物の存在を確認するために40倍の倍率で光学顕微鏡を使用しています。細胞培養プレートからすべてのRPMI培地を除去し、穏やかに50 mLの血清学的ピペットを使用して、リン酸緩衝生理食塩水（PBS）で2回接着細胞を洗浄します。 注：脂質体はTHP-Mの細胞質のように、小さい透明な球状構造で表示されます。 </li><li>各プレートに、PBS中の5mMのエチレンジアミン四酢酸（EDTA）の40ミリリットルを加えます。 37°Cで15分間インキュベートします。 </li><li>繰り返しピペッティング全体板の表面上に10-mLの血清学的ピペットを使用して、泡沫状マクロファージ（FM）を取り外します。 50 mLコニカルチューブに細胞懸濁液を移し、5分間、150×gでスピンダウン。 </li><li> 10 mlのPBS（第三のPBS洗浄）Aで細胞ペレットを再懸濁予め計量した15mLコニカルチューブに移す。 150xgで5分間再びスピンダウンする。血清学的ピペットを使用して上清を慎重に吸引し、廃棄する。 </li><li> FMペレットを3回凍結融解して細胞を溶解し、75℃で20〜30分間インキュベートしてマトリックス中のタンパク質を変性させます。使用準備が整うまでペレットを-20℃で保存する。 </li></ol>迅速な平衡透析（RED）アッセイ<ol><li>ジメチルスルホキシド（DMSO）中のすべての試験化合物の10 mMストック溶液を調製する。各アッセイの前にDMSO中で500μMの作業溶液に希釈する。 </li><li>カゼインサロゲートペレットを入れたチューブを秤量する。空のチューブの重量を引いて、ペレットのみの重量を導出する。チューブ当たり2〜3個の金属ビーズを添加し、組織ホモジナイザーを用いて1,200ストローク/分で1分間、各マトリックスの10倍希釈懸濁液を達成するために、カセムまたは代理マトリックスをPBS（1：9w / v） </li><li>スパイク6.5μLの500μL5μM（≦1％DMSO）およびボルテックスの最終濃度を達成するために、643.5μLのホモジネートに試験化合物のμM溶液を添加する。 </li><li> REDインサートをベースプレートに挿入します。各REDインサートのドナーチャンバー（赤いリング）と各レシーバーチャンバーへの350μLのPBSに200μLの薬物添加マトリックスを加えます。各試験化合物について3つのインサートを調製する（3つのサンプル）。プレートを接着プレートシールでシールし、サーモミキサー上で37℃、200 rpm（1 xg）で4時間インキュベートする。 </li><li>インキュベーション後、ドナーおよびレシーバーチャンバーの内容物を2〜3回上下にピペッティングすることにより穏やかに混合する。ドナーチャンバーからホモジネートのアリコート20μLをピペットで出し、1.5mLチューブ（1：1）中の20μLのきれいなPBSに加える。同様に、レシーバーチャンバーからのPBSサンプルのアリコート20μLをピペットで取り出し、クリーンホモジネート（マトリックスマッチング）20μLを加えます。 8 注：マトリックスマッチングは、定量分析のために2つの別々の較正曲線（ホモジネートおよびPBS中）を作成した。ドナーチャンバーの内容物は時間の経過とともに沈降することがある。アリコートを除去する前にピペットで内容物を穏やかに混合する。 </li></ol> 4.LC-MS定量およびデータ解析<ol><li> 500ng / mLのジクロフェナクまたは10ng / mLのベラパミル（内部標準）を含む160μLの1：1メタノール：アセトニトリルを各チューブに加え、ボルテックスしてタンパク質を沈殿させる。 10,000×gで5分間遠心分離して沈殿物を沈降させ、液体クロマトグラフィー - 質量分析（LCMS）分析のために上清を96ウェルディープウェルプレートに移す。 7 </li><li>上記のサンプルと同じマトリックス組成を維持しながら、各試験化合物について1〜1,000nMの較正曲線を構築する。 LC-MS法を用いて、ドナーおよびレシーバーチャンバーからのサンプル中の試験化合物の濃度を定量する。 </li><li>非結合部分（ f u ）を計算する式1を用いて希釈マトリックス中の薬物を計算する。式2を用いて希釈されていないマトリックス中のf uを計算する（ D =希釈係数10）。 14 <img alt="式1" src="/files/ftp_upload/55559/55559eq1.jpg" /> <img alt="式2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li><li>式3を用いて各化合物の回収（質量バランス）を調べ、安定性/代謝/非特異的結合の問題を有する化合物を同定する。 注：回復率は通常70〜130％です。 15 <img alt="式3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li></ol>

<ol>
	<li>Sacchettini, J. C., Rubin, E. J., Freundlich, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+versus+bugs:+in+pursuit+of+the+persistent+predator+Mycobacterium+tuberculosis.">Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis.</a> Nat Rev Microbiol. 6 (1), 41-52 (2008).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+and+dormant+tubercle+bacilli+and+latent+tuberculosis.">Persistent and dormant tubercle bacilli and latent tuberculosis.</a> Front Biosci. 1 (9), 1136-1156 (2004).</li><li>Aber, V. R., Nunn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+term+chemotherapy+of+tuberculosis.+Factors+affecting+relapse+following+short+term+chemotherapy.">Short term chemotherapy of tuberculosis. Factors affecting relapse following short term chemotherapy.</a> Bull Int Union Tuberc. 53 (4), 276-280 (1978).</li><li>Chang, K. C., Leung, C. C., Yew, W. W., Ho, S. C., Tam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nested+case-control+study+on+treatment-related+risk+factors+for+early+relapse+of+tuberculosis.">A nested case-control study on treatment-related risk factors for early relapse of tuberculosis.</a> Am J Respir Crit Care Med. 170 (10), 1124-1130 (2004).</li><li>Dartois, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+of+anti-tuberculosis+drugs:+from+blood+to+lesions+to+mycobacterial+cells.">The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells.</a> Nature Rev Microbiol. 12 (3), 159-167 (2014).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+sterilizing+activity+and+drug+distribution+into+tuberculosis+lesions.">The association between sterilizing activity and drug distribution into tuberculosis lesions.</a> Nat Med. 21 (10), 1223-1227 (2015).</li><li>Sarathy, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+Drug+Penetration+in+Tuberculosis+Lesions.">Prediction of Drug Penetration in Tuberculosis Lesions.</a> ACS Infect Dis. 2 (8), 552-563 (2016).</li><li>Waters, N. J., Jones, R., Williams, G., Sohal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+rapid+equilibrium+dialysis+approach+for+the+measurement+of+plasma+protein+binding.">Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding.</a> J Pharm Sci. 97 (10), 4586-4595 (2008).</li><li>Singh, J. K., Solanki, A., Maniyar, R. C., Banerjee, D., Shirsath, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Equilibrium+Dialysis+(RED):+an+In-vitro+High-Throughput+Screening+Technique+for+Plasma+Protein+Binding+using+Human+and+Rat+Plasma.">Rapid Equilibrium Dialysis (RED): an In-vitro High-Throughput Screening Technique for Plasma Protein Binding using Human and Rat Plasma.</a> J Bioequiv Availab. 14, 1-4 (2012).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbound+drug+concentration+in+brain+homogenate+and+cerebral+spinal+fluid+at+steady+state+as+a+surrogate+for+unbound+concentration+in+brain+interstitial+fluid.">Unbound drug concentration in brain homogenate and cerebral spinal fluid at steady state as a surrogate for unbound concentration in brain interstitial fluid.</a> Drug Metab Dispos. 37 (4), 787-793 (2009).</li><li>Able, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+localization,+native+tissue+binding+and+ex+vivo+occupancy+for+centrally+penetrant+P2X7+antagonists+in+the+rat.">Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat.</a> Br J Pharmacol. 162 (2), 405-414 (2011).</li><li>Subbian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+pulmonary+cavitary+tuberculosis+in+rabbits:+a+failed+host+immune+response.">Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response.</a> Open Biol. 1 (4), 1-14 (2011).</li><li>Via, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculous+Granulomas+are+Hypoxic+in+Guinea+pigs,+Rabbits,+and+Non-Human+Primates.">Tuberculous Granulomas are Hypoxic in Guinea pigs, Rabbits, and Non-Human Primates.</a> Infect Immun. 76 (6), 2333-2340 (2008).</li><li>Kalvass, J. C., Maurer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+nonspecific+brain+and+plasma+binding+on+CNS+exposure:+implications+for+rational+drug+discovery.">Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery.</a> Biopharm Drug Dispos. 23 (8), 327-338 (2002).</li><li>Di, L., Umland, J. P., Trapa, P. E., Maurer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+recovery+on+fraction+unbound+using+equilibrium+dialysis.">Impact of recovery on fraction unbound using equilibrium dialysis.</a> J Pharm Sci. 101 (3), 1327-1335 (2012).</li><li>Lenaerts, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+persisting+mycobacteria+in+a+Guinea+pig+model+of+tuberculosis+revealed+by+r207910.">Location of persisting mycobacteria in a Guinea pig model of tuberculosis revealed by r207910.</a> Antimicrob Agents Chemother. 51 (9), 3338-3345 (2007).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+MALDI-MRM-MS+imaging+of+moxifloxacin+distribution+in+tuberculosis-infected+rabbit+lungs+and+granulomatous+lesions.">High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.</a> Anal Chem. 83 (6), 2112-2118 (2011).</li></ol>

競合する金銭的利益はない。

結核に感染した患者の肺壊死病変および空洞には、薬物治療に対して反抗的な細菌の亜集団が含まれる。これらの構造の大部分のコアは、細胞外環境においてこれらの胞子を保持することに特に関与する。これらの離れた場所への抗菌剤の好都合な分布は、結核薬効の重要な決定要因であると考えられている。このプロトコールのバリデーションの前に、大腸病変における創薬化合物の分布?...

肺結核疾患の治療には、様々なタイプの病変への薬物の効果的な分配が必要である。壊死病巣および腔は、薬剤耐性または「持続性」細菌の亜集団を有する大腸菌センターを含む。 1、2空洞病は、劣悪な治癒率と不良な予後と関連している。以前の研究では、定量的および画像技術を用いて、カゼムを浸透させる能力が薬物クラスごとに著しく異なることが示されている。しかし、これらの方法は、遅く退屈な動物感染モデルの使用を必要とする。 ex vivoカゼウムへの薬物結合を測定するインビトロアッセイを設計した。この結合は、肉芽腫の薬物浸透と逆相関することが判明し、したがって、予測ツールとして使用。 7 平衡透析は、血漿蛋白質結合研究に金標準的なアプローチとみなされています。迅速平衡透析（RED）デバイスは、そのようなアッセイを実施するための、迅速で使いやすいと信頼性の高いシステムを提供します。 8デバイスは、2つのコンポーネントで構成され：使い捨て、半透膜の垂直シリンダによって分離された2室からなる使い捨てインサート。一度に48個のインサートまで保持することができ、再使用可能なベース板。透析膜は、8 kDaの分子量カットオフ（MWCO）を有する薬物 - 高分子結合研究のために理想的です。膜区画の高い表面積対体積比は、急速透析および平衡化を可能にします。インサートとベースプレートの両方は、最小の非特異的結合のために検証されてきました。生体分析技術とRED素子の組み合わせは、Pにおける薬物の非結合画分の正確な推定を提供しますラスマ8,9 当初は血漿タンパク質結合を測定するように設計されていたが、RED装置はホモジネートを用いたいくつかの組織結合研究に用いられてきた。このプロトコールでは、壊死病変および結核感染ウサギの腔から摘出された壊死屑であるカゼムに対する薬物結合を測定する。ケース状材料の無細胞性および非血管性は、アッセイと適合する均一な懸濁液にホモジナイズすることを容易にする。 カゼウムが生産するのが面倒で、来るのが難しいことを考えると、プロトコールはまた、泡沫状マクロファージから調製された代理母材と共に使用するために検証されている。 THP-1単球由来マクロファージはオレイン酸で誘導され、それらに「泡沫」外観を与える複数の脂質体を蓄積する。これらの脂質負荷細胞を採取し、我々はcaseumへの代理として使用マトリックスを生成するために処理。この研究では、この代用マトリックスに結合するその薬剤を示した効果肉芽腫及び空洞の乾酪性コア中の薬物の浸透を妨げるインビボのプロセスを模倣、caseumに結合するとよく相関します。

<table border="0" cellpadding="0" cellspacing="0" width="960">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">New Zealand White Rabbits</td>
			<td style="width:203px;">Covance</td>
			<td style="width:147px;">-</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:203px;">BEI Resources</td>
			<td style="width:147px;">NR-13647&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ketathesia (Ketamine) 100mg/mL C3N</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">56344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Anased (Xylazine) 100mg/mL</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">33198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:203px;">Virbac</td>
			<td style="width:147px;">710101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">THP-1 monocytic cell line</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:147px;">ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">175cm&#178; TC-Treated Flask (T175)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-3400-175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 media w/o glutamine</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>MT-15-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone Fetal Bovine Serum, Gamma irradiated&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3091003IR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone L-glutamine, 200mM</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellstar TC dish, 145x20mm, vented</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-2881-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate13-acetate (PMA)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>BP685-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:203px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>ICN15178101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Reusable Base Plate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Inserts, 50/box&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED insert removal tool&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>89812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive plate seal</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH7.4, 10 X 500 mL (Gibco)&#160;</td>
			<td style="width:203px;">Life Technologies&#160;</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">DMSO</td>
			<td style="width:203px;">Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Acetonitrile</td>
			<td style="width:203px;">Sigma</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Methanol</td>
			<td style="width:203px;">Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Verapamil hydrochloride</td>
			<td style="width:203px;">Sigma</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Diclofenac sodium salt</td>
			<td style="width:203px;">Sigma</td>
			<td>93484</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ethanol, 200 proof</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">2010 Geno/Grinder</td>
			<td style="width:203px;">SPEX SamplePrep</td>
			<td style="width:147px;">2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Bead Mill Homogenizer Accessory, Metal Bulk Beads</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-340-158</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">484R Cobalt 60 Irradiator</td>
			<td style="width:203px;">JL Shepard</td>
			<td style="width:147px;">7810-484-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">INCYTO&#160;C-Chip Disposable Hemacytometers</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:147px;">22-600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Upright Light Microscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:147px;">DM1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Binary Liquid Chromatography system</td>
			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Mass spectrometer</td>
			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro caseum binding assay that predicts drug penetration in tuberculosis lesions

結核病の根絶には、複雑な肺病変の複数の層に浸透することができる薬物レジメンが必要である。窩洞および病変の大部分のコアにおける薬物分布は、一般に害虫とも呼ばれる薬物耐性細菌の亜集団を収容するため、特に重要である。結核病変における薬剤浸透の測定のための既存の方法は、 生体分析または画像化技術に結合された経費のかかる時間のかかる生体内薬物動態研究を必要とする。この結合は、カゼムを介する薬物分子の受動的拡散を妨げるので、カゼイン高分子への薬物結合のインビトロ測定は、そのような技術の代替として提案されている。迅速な平衡透析は、血漿タンパク質および組織結合研究を行うための迅速かつ信頼性の高いシステムである。このプロトコールでは、excisであるカゼウムのホモジネートへの薬物結合を測定するために、高速平衡透析（RED）装置を使用した結核に感染したウサギの病変および腔から滲出した。このプロトコールはまた、カゼムの代わりに使用する脂質負荷THP-1マクロファージから代理マトリックスを生成する方法も記載している。このカゼウム/サロゲート結合アッセイは、結核薬の発見において重要なツールであり、他の疾患によって引き起こされる病変または膿瘍の薬物分布を研究するのに役立つことができる。

このプロトコルを使用して、我々は浸透カゼムでの予測された効率のために何百もの結核薬開発化合物を試験した。 図1は、REDアッセイの基本概念を視覚化しています。 REDインサートの透析膜は、未結合の小分子がドナーウェルからレシーバウェルに拡散し、最終的に両方のコンパートメント間の平衡を達成することを可能にする。タンパク質または脂?...

Watch this Scientific Journal Video about An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions at JoVE.com

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

ここでは、肺結核病変および腔からのカゼムとの薬物結合を測定するための迅速な平衡透析（RED）法を説明する。このプロトコールはまた、発泡マクロファージ由来のマトリックスと共に使用され、これは、カゼムの有効な代用物である。

アン<em&gt;インビトロ</em&gt;結核病変における薬物侵入を予測するカゼウム結合アッセイ

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

an-vitro-caseum-binding-assay-that-predicts-drug-penetration

Research

Education

JoVE Journal

JoVE Core

Pharmacokinetics and Pharmacodynamics

Pharmacology

Immunology and Infection

Unit 1

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nonlinear Pharmacokinetics

Introduction to Respiratory System Drugs

SCIENTISTS IN ACTION

11.6K ビュー.  Rutgers. ここでは、肺結核病変および腔からのカゼムとの薬物結合を測定するための迅速な平衡透析（RED）法を説明する。このプロトコールはまた、発泡マクロファージ由来のマトリックスと共に使用され、これは、カゼムの有効な代用物である。 

ビデオ: An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8ウェルチャンバースライドを用いたin vitro のバイオフィルム形成における

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

免疫・感染学

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

ゼブラフィッシュで自動化されたハイコンテンツ炎症アッセイ：創薬を促進する

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

自然送信時に結核マラリア重複感染を研究する実験モデル結核菌とマラリアベルゲイ</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用肺炎球菌植民に対するプロバイオティクスの影響を調査<em&gt;体外</em&gt;接着アッセイ

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

ダルババンシンの参照ブロス微量希釈法<em&gt;インビトロ</em好気的に成長する細菌の&gt;感受性試験

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

調節するc-ジ-GMPシグナリングで、小さな分子をスクリーニングするためのハイスループットセットアップの確立<em&gt;緑膿菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

マクロファージ継代に対する薬効を評価するために、ハイスループットアッセイの互換性<em&gt;結核菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

治療抗体間の標的結合およびCDC誘導を比較するためのインビトロ法：Biosimilarity Analysisにおける応用

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...