SZ e DW ricevono una sovvenzione illimitata di Viropharma.

1. Preparazione di bromelina trattati globuli rossi <ol><li> Globuli rossi Lavare 0 tipizzate 3x con 1x PBS (centrifugazione a 664 xg per 2-5 min). </li><li> Incubare 1 volume di eritrociti concentrati con due volumi di una soluzione bromelina 0,5% per 10 min a 37 ° C. </li><li> Lavare le cellule 3x 1x PBS. </li><li> Le cellule possono essere memorizzati come una soluzione al 3% a 4 ° C in PBS 1x per almeno una settimana. </li></ol> 2. C3 e C4 Deposizione Analisi su globuli rossi di FACS <ol><li> Calore inattivare la quantità necessaria di siero del paziente (o altra sorgente anticorpo anti-RBC) mediante riscaldamento del campione per 30 minuti a 56 ° C. </li><li> Lavare RBC bromelina trattati tre volte in VBG - (5 veronal mM, NaCl 150 mM, 0,05% gelatina pH 7.4) e risospendere in VBG + + (VBG - + 2 mM MgCl 2 + 10 mM CaCl 2) per dare un soluzione allo 0,5%. </li><li> Pipettare i seguenti componenti in un piatto fondo rotondo con una perla di vetro (fo corretta miscelazione): 25 microlitri 0,5% globuli rossi, 37,5 ml di siero fresco AB (fonte complemento) e 37,5 ml 200 mg / ml anti-C5. Anti-C5 è costoso e potrebbe essere difficile da ottenere. Si potrebbe considerare l&#39;utilizzo di siero C5-/C6-/C7- o C8-deficienti, invece di siero AB e anti-C5. </li><li> Aggiungere siero del paziente per pozzetto per una concentrazione finale di 0,1-33%. Corretto per le differenze di concentrazione sierica per pozzetto dovute utilizzando siero AB inattivato al calore. Aggiungere VBG + + per ottenere un volume finale di 150 microlitri / pozzetto. Chiudi piastra con ELISA pellicola. </li><li> Incubare 1,5-2 ore a 37 ° C sotto agitazione. </li><li> Lavare gli RBC tre volte con PBS + 0,5% BSA (centrifugazione a 664 xg per 2 minuti). </li><li> Aggiungere fluorescente anti-C3 e anticorpi monoclonali anti-C4 o loro frammenti Fab (per ridurre l&#39;agglutinazione) in PBS + 0,5% BSA ad una concentrazione finale di 1 mg / ml. Qui personalizzata sviluppata anti-C3-Alexa Fluor 488 e vengono utilizzati anti-C4-Alexa Fluor 647 anticorpi monoclonali, la scelta di These le etichette fluorescenti permette la rilevazione simultanea di C3 e C4 da FACS. </li><li> Incubare 30-45 minuti a temperatura ambiente agitando delicatamente. </li><li> Lavare globuli rossi 3x con PBS + 0,5% BSA. </li><li> Risospendere GR 150 microlitri di PBS +0,5% BSA e li pipetta in una nuova piastra (di sbarazzarsi delle perle di vetro prima dell&#39;analisi FACS). </li><li> Eseguire l&#39;analisi FACS. Separare singole cellule da doppietti in una dispersione tracciato FSC-A, impostare la porta sui singoli globuli rossi; selezionare il canale di rilevamento appropriato per i segnali di fluorescenza (ad esempio FITC e APC). </li><li> Quantificare risultati usando l&#39;intensità di fluorescenza mediana. </li></ol> Nota: E &#39;possibile utilizzare un controllo isotipico (es fluorescente anti-IL6 topo IgG1). Tuttavia, si raccomanda di includere un vero controllo negativo esempio incubando RBC senza siero del paziente (solo siero AB) o senza una fonte complemento (AB siero inattivato al calore e siero inattivato al calore), und poi macchiare questi con lo stesso anti-C3-Alexa Fluor 488 e anti-C4-Alexa Fluor 647 anticorpi. Questo dà un controllo più valido di un isotipo di controllo in caso di globuli rossi 12, sebbene questi diversi controlli tipicamente danno lo stesso, esito negativo. 3. Quantitative Assay emolitica <ol><li> Calore inattivare la quantità necessaria di sieri dei pazienti (o altra sorgente anticorpo anti-RBC) riscaldando il campione 30 min a 56 ° C. </li><li> Lavare globuli rossi 3x con VBG - e una volta con VBG + +. </li><li> Pipettare i seguenti componenti in un piatto fondo rotondo con una perla di vetro (per una corretta miscelazione): 35 ml 3% globuli rossi, 25 ml di siero fresco AB (fonte complemento) e il siero del paziente 1-50%. Corretto per le differenze di concentrazione del siero utilizzando siero AB inattivato al calore. Aggiungere VBG + + per ottenere un volume finale di 150 microlitri / pozzetto. Chiudi piastra con ELISA pellicola. </li><li> Incubare a 37 ° C per 1,5-2 ore con agitazione. </li><li> CPiastra entrifuge a 664 xg per 5 minuti con la decelerazione ridotta (ad esempio, la decelerazione alla piena fermata in 2-3 min). </li><li> Pipetta con cautela 90 microlitri surnatante in una nuova micropiastra. È importante, in questa fase per evitare bolle d&#39;aria e per evitare di portare con cellule intatte. </li><li> Misurare A414/690 in uno spettrofotometro adeguato. </li><li> Esprimere la lisi come percentuale della lisi di un campione eritrociti con acqua pura. </li></ol>

<ol>
	<li>Zeerleder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+haemolytic+anaemia+-+a+practical+guide+to+cope+with+a+diagnostic+and+therapeutic+challenge.">Autoimmune haemolytic anaemia - a practical guide to cope with a diagnostic and therapeutic challenge.</a> Neth. J. Med. 69 (4), 177-184 (2011).</li><li>Reardon, J. E., Marques, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+evaluation+and+transfusion+support+of+patients+with+autoimmune+hemolytic+anemia.">Laboratory evaluation and transfusion support of patients with autoimmune hemolytic anemia.</a> Am. J. Clin. Pathol. 125, 71-77 (2006).</li><li>Lai, M., Leone, G., Landolfi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+hemolytic+anemia+with+gel-based+immunohematology+tests.">Autoimmune hemolytic anemia with gel-based immunohematology tests.</a> Am. J. Clin. Pathol. 139 (4), 457-463 (2013).</li><li>Zantek, N. D., Koepsell, S. A., Tharp Jr, ., R, D., Cohn, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+direct+antiglobulin+test:+A+critical+step+in+the+evaluation+of+hemolysis.">The direct antiglobulin test: A critical step in the evaluation of hemolysis.</a> Am. J. Hematol. 87 (7), 707-709 (2012).</li><li>Natukunda, B., Brand, A., Schonewille, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+alloimmunization+from+an+African+perspective.">Red blood cell alloimmunization from an African perspective.</a> Curr. Opin. Hematol. 17 (6), 565-570 (2010).</li><li>Packman, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+anemia+due+to+warm+autoantibodies.">Hemolytic anemia due to warm autoantibodies.</a> Blood Rev. 22 (1), 17-31 (2008).</li><li>Berentsen, S., Tj&#248;nnfjord, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+cold+agglutinin+mediated+autoimmune+hemolytic+anemia.">Diagnosis and treatment of cold agglutinin mediated autoimmune hemolytic anemia.</a> Blood Rev. 26 (3), 107-115 (2012).</li><li>Petz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold+antibody+autoimmune+hemolytic+anemia.">Cold antibody autoimmune hemolytic anemia.</a> Blood Rev. 22 (1), 1-15 (2008).</li><li>Barros, M. M. O., Blajchman, M. A., Bordin, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warm+autoimmune+hemolytic+anemia:+recent+progress+in+understanding+the+immunobiology+and+the+treatment.">Warm autoimmune hemolytic anemia: recent progress in understanding the immunobiology and the treatment.</a> Transfus. Med. Rev. 24 (3), 195-210 (2010).</li><li>Endoh, T., 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=Optimal+prewarming+conditions+for+Rh+antibody+testing.">Optimal prewarming conditions for Rh antibody testing.</a> Transfusion. 46 (9), 1521-1525 (2006).</li><li>Wouters, D., 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=C1-esterase+inhibitor+concentrate+rescues+erythrocytes+from+complement-mediated+destruction+in+autoimmune+hemolytic+anemia.">C1-esterase inhibitor concentrate rescues erythrocytes from complement-mediated destruction in autoimmune hemolytic anemia.</a> Blood. 121 (7), 1242-1244 (2013).</li><li>Arndt, P. A., Garratty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Review+of+Published+Methods+for+Analysis+of+Red+Cell+Antigen-Antibody+Reactions+by+Flow+Cytometry,+and+Approaches+for+Resolving+Problems+with+Red+Cell+Agglutination.">A Critical Review of Published Methods for Analysis of Red Cell Antigen-Antibody Reactions by Flow Cytometry, and Approaches for Resolving Problems with Red Cell Agglutination.</a> Transfus. Med. Rev. 24 (3), 172-194 (2010).</li></ol>

Gli autori non hanno nulla da rivelare.

I dosaggi di cui sopra sono riproducibili e robusto. Essi sono di facile esecuzione ed è possibile effettuare loro con molti campioni Allo stesso tempo (in una piastra a 96 pozzetti). Pertanto, questo approccio sarebbe idoneo per un sistema completamente automatico, ad esempio un sistema robot ELISA. Contrariamente alle tecniche attualmente utilizzate, questi test sono quantitative e questo permette, ad esempio nello studio degli effetti di inibitori del complemento. Inoltre, l&#39;interpretazione è ...

Gli anticorpi contro i globuli rossi (RBC) può essere indotta da trasfusione di globuli rossi che esprimono un antigene che non è presente in globuli rossi del destinatario. Questi anticorpi ALLO possono causare gravi reazioni trasfusionali emolitiche acute dovute a completare l&#39;attivazione sul seguente trasfusione 5. In auto-immune anemia emolitica (MEA), i pazienti hanno auto-anticorpi contro i propri globuli rossi. Questo porta alla clearance accelerata delle cellule attraverso l&#39;interazione di IgG legati a globuli rossi con Fcy-recettori sulla fagociti nella milza e / o in caso di auto-anticorpi capaci di attivare il complemento attraverso recettori del complemento nel fegato 6,7. Attivazione del complemento fulminante con conseguente emolisi intravascolare è rara, ma spesso fatale. Accelerata, integrare mediata distruzione RBC indotta da entrambi risultati-allo o autoanticorpi in anemia acuta e ipossia tissutale, quindi, potenzialmente fatale. Gli auto-anticorpi in MEA sono classificati in anticorpi caldi e freddi, depending della temperatura ottimale si legano a RBC (rispettivamente 37 ° C o inferiori,). Gli anticorpi caldi sono generalmente di isotipo IgG e gli anticorpi fredde del isotipo IgM 8,9. MEA può essere secondaria ad es lymphoprolyferative disturbi, malattie del tessuto connettivo, tumori solidi, infezioni o droghe, ma nel 50% dei casi è idiopatica MEA 9. Rilevamento di gammaglobuline (ad es. IgG o IgM) e complementare legato a globuli rossi del paziente viene eseguita mediante un diretto all&#39;antiglobulina semiquantitativa (Coombs) Test (DAT). Nella DAT globuli rossi del paziente vengono incubati con anticorpi anti-IgG o anti-C3d. Presenza di RBC agglutinazione dimostra la presenza di componenti del complemento montati o IgG vincolante. Rilevamento di-allo o autoanticorpi nel siero del paziente viene eseguita mediante il test indiretto (IAT). Nel IAT, globuli rossi prova bromelina trattati vengono incubati con siero del paziente, lavate e poi incubate con anti-hUman IgG. Nel caso in cui i globuli rossi sono stati sensibilizzati con anticorpi anti-IgG RBC presenti nel agglutinazione siero del paziente si verificherà. Anticorpi IgM anti globuli rossi saranno direttamente portare a agglutinazione su di incubazione di globuli rossi prova bromelina trattati con siero del paziente. RBC agglutinazione nel test di Coombs diretto o in IAT è valutata visivamente sia dall&#39;occhio in una provetta o caricando il campione su una piccola colonna Sephadex separa RBC agglutinate e singoli per dimensione 1. Un&#39;altra tecnica frequentemente utilizzata per misurare l&#39;attivazione del complemento su globuli rossi è il saggio emolitico 1, in cui viene valutata la capacità del siero del paziente per indurre (complemento-mediata) emolisi dei globuli rossi bromelina trattati 10. Il test è notato come positivo quando il surnatante dopo centrifugazione è macchiato rosso causa emoglobina rilasciata e considerato negativo se rimane incolore. Sia il test antiglobulina e il saggio emolitico sono semiquantitativa, poiché la massima Serum diluizione è indicato in cui il test è ancora positivo. Il test di Coombs e il saggio emolitico sono test affidabili che sono abitualmente utilizzati nella diagnostica. Poiché questi saggi sono semiquantitativa e dipendente dall&#39;esperienza del tecnico esecuzione del saggio non sono adatti per studiare le sottili differenze di attivazione del complemento di globuli rossi, come necessario quando valutare l&#39;efficacia di inibitori del complemento. Pertanto, abbiamo sviluppato due analisi quantitative per determinare l&#39;attivazione del complemento da anticorpi (auto-) per i globuli rossi, che descriveremo in questo articolo. In primo luogo, abbiamo sviluppato un saggio per misurare la deposizione di frammenti attivazione del complemento C3 e C4 su globuli rossi (Figura 1A). In questo saggio, bromelina trattati umane di tipo-0 globuli rossi vengono incubati con siero inattivato al calore del paziente (anti-RBC fonte anticorpi), siero fresco AB (fonte complemento) e anti-C5 anticorpo monoclonale (Eculizumab). Durante questa incubation, C3 e C4 deposizione si verifica se il siero del paziente contiene completano l&#39;attivazione di anticorpi anti-RBC. Per evitare di lisi da attivazione del complemento a valle viene aggiunto un anticorpo monoclonale anti-C5 blocco. Successivamente, C3 e C4 deposizione sul globuli rossi viene rilevato mediante FACS utilizzando anticorpi monoclonali fluorescente o frammenti Fab-reagiscono con C3 e C4, rispettivamente. Gating sul singolo globuli rossi è importante per garantire l&#39;affidabilità dei risultati. I vantaggi di questa tecnica sono che è necessaria una piccola quantità di materiale paziente, l&#39;attivazione del complemento in una fase iniziale della cascata è valutato e il metodo è riproducibile e quantitativa. Un ulteriore vantaggio di guardare sia C3 e C4 è che una distinzione può essere fatta tra l&#39;attivazione classica e lectina pathway (sia C3 e C4 deposizione) e l&#39;attivazione della via alternativa (solo C3 deposizione). L&#39;uso di GR bromelina trattato invece di eritrociti non trattati aumenta la sensibilità del comedire. Il secondo saggio è basato sul dosaggio emolitica attualmente utilizzato (Figura 1B). Umani bromelina trattati tipo 0 globuli rossi vengono incubati con siero inattivato al calore del paziente (anti-RBC fonte anticorpi) e siero fresco AB (fonte complemento). Se complemento è attivato da anticorpi anti-RBC paziente, si verificherà dose-dipendente di lisi, porta al rilascio di emoglobina. La quantità di emoglobina rilasciata viene quantificato misurando la sua assorbanza a 414 nm nel supernatante dopo la filatura le eritrociti intatti e frammentati. L&#39;assorbanza è correlata alla quantità di emolisi verificato. In contrasto con il dosaggio attualmente in uso, questo protocollo permette un obiettivo, valore quantitativo di emolisi che è molto riproducibile e non dipende dalla persona interpretazione del saggio. Un&#39;applicazione di questi metodi è stato descritto in 11, in cui l&#39;uso potenziale di C1-inibitore come inibitore del complemento in MEA era studied.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:195px;">Fresenius Kabi</td>
			<td style="width:119px;"></td>
			<td style="width:316px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">B-4287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0261</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Barbital</td>
			<td style="width:195px;">Fagron</td>
			<td style="width:119px;">0263</td>
			<td>This chemical is subject to drug regulation.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelatin</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0470.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.06404.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium chloride</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">1.0238.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 488 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">46402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dylight 650 amine reactive dye</td>
			<td style="width:195px;">Pierce</td>
			<td style="width:119px;">62265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#945;C5 (Eculizumab)</td>
			<td style="width:195px;">Alexion Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS (Canto)</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;"></td>
			<td>Any FACS can be used that has the appropiate lasers.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spectrophotometer (e.g. Multiskan spectrum)</td>
			<td style="width:195px;">Thermo Labsystems</td>
			<td style="width:119px;">1500-193</td>
			<td>Any spectrophotometer with the right wavelength range can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD FACSDiva software v 6.1.2</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">643629</td>
			<td>Any compatible FACS analysis software can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bromelain</td>
			<td style="width:195px;">Sanquin</td>
			<td style="width:119px;">K1121</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for quantitative detection of antibody-induced complement activation on red blood cells

Gli anticorpi contro i globuli rossi (RBC) possono portare ad attivazione del complemento con un conseguente approvazione accelerata attraverso recettori del complemento nel fegato (emolisi extravascolare) o portano alla lisi intravascolare dei globuli rossi. Alloanticorpi (ad esempio ABO) o gli anticorpi degli antigeni RBC (come si è visto in anemia emolitica autoimmune, MEA) che portano alla attivazione del complemento sono potenzialmente dannose e possono essere - soprattutto quando conduce alla lisi intravascolare - fatale 1. Attualmente, l&#39;attivazione del complemento a causa di (auto-anticorpi) in globuli rossi viene valutata in vitro utilizzando il test di Coombs riflettendo complemento deposizione su RBC o da un saggio emolitico nonquantitative riflettendo RBC lisi 1-4. Tuttavia, per valutare l&#39;efficacia di inibitori del complemento, è obbligatorio avere tecniche quantitative. Qui descriviamo due tali tecniche. In primo luogo, un test di rilevare C3 e C4 deposizione su globuli rossi che è indotto da anticorpi nel siero del paziente è prepresentate. Per questo, analisi FACS è usato con anticorpi anti-C3 o anti-C4 fluorescente. Accanto, un saggio emolitico quantitativo è descritto. Emolisi In questo saggio, complemento-mediata indotta dal siero del paziente viene misurata facendo uso di rilevazione spettrofotometrica della emoglobina rilasciata. Entrambi questi saggi sono molto riproducibile e quantitativa, facilitando studi di attivazione del complemento anticorpo-indotta.

Figura 2A mostra un grafico rappresentante dispersione di globuli rossi. Gating Adatto per singoli globuli rossi può essere visto in de FSC-W - FSC-A plot (P1). Solitamente circa il 95% dei globuli rossi rientrano in questa porta P1 (singole cellule), ma quando viene usata una percentuale di siero del paziente, questo può cadere al 70-80%, specialmente quando anti-RBC IgM è presente nel siero del paziente. Risultati rappresentativi per l&#39;effetto del siero del paziente...

Watch this Scientific Journal Video about Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells at JoVE.com

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Qui si descrivono due saggi per misurare l&#39;attivazione del complemento indotta da anticorpi contro le cellule rosse del sangue. Il principale vantaggio rispetto ai test attuali è la loro da interpretare facile natura quantitativa e.

Metodi per quantitativa individuazione di anticorpi indotta da attivazione del complemento sui globuli rossi

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

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Immunology and Infection

30.4K Views.  University of Amsterdam.  Qui si descrivono due saggi per misurare l'attivazione del complemento indotta da anticorpi contro le cellule rosse del sangue. Il principale vantaggio rispetto ai test attuali è la loro da interpretare facile natura quantitativa e. 

Video: Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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Immunologia e infezioni

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

Isolamento di cellule della cavità peritoneale mouse

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modello murino di CD40-attivazione delle cellule B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Rilevazione di attività NEU1 sialidasi nella regolamentazione Toll-like l&#39;attivazione del recettore

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Protocollo per ricombinante RBD-based Vaccines SARS: Preparazione di proteine, vaccinazione degli animali e la rivelazione di neutralizzazione

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Gli antigeni Protetto funzionali globuli rossi dalla membrana innesto di Compact Poligliceroli Hyperbranched

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Saggi qualitativi e quantitativi per il rilevamento e la caratterizzazione di proteine ​​antimicrobici

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...