SZ et DW reçoivent une subvention sans restriction de Viropharma.

Une. Préparation des globules rouges broméline traités <ol><li> Globules rouges Laver 0 typées 3x avec 1x PBS (centrifugation à 664 g pendant 2-5 min). </li><li> Incuber 1 volume de globules rouges emballées avec deux volumes d&#39;une solution de broméline à 0,5% pendant 10 min à 37 ° C. </li><li> Laver les cellules 3x avec PBS 1x. </li><li> Les cellules peuvent être stockées sous la forme d&#39;une solution à 3% à 4 ° C dans 1 x de PBS pendant au moins une semaine. </li></ol> 2. C3 et C4 dépôt analyse des globules rouges par FACS <ol><li> Inactiver à la chaleur la quantité requise de sérum d&#39;un patient (ou une autre source d&#39;anticorps anti-RBC) en chauffant l&#39;échantillon pendant 30 min à 56 ° C. </li><li> Laver les hématies de bromélaïne traitée trois fois dans VBG - (5 mM véronal, NaCl 150 mM, 0,05% de gélatine, pH 7,4) et les remettre en suspension dans VBG + + (VBG - + 2 mM de MgCl2 + 10 mM de CaCl2) pour donner un solution à 0,5%. </li><li> Introduire à la pipette les éléments suivants dans une plaque à fond rond avec une perle de verre (fou un mélange approprié): 25 pi 0,5% de globules rouges, 37,5 ul de sérum frais AB (en complément) et 37,5 pi 200 pg / ml anti-C5. Anti-C5 est cher et peut être difficile à obtenir. On pourrait envisager d&#39;utiliser du sérum C5-/C6-/C7- ou C8 déficient au lieu de sérum AB et anti-C5. </li><li> Ajouter le sérum du patient par puits à une concentration finale de 0,1 à 33%. Corriger les différences de concentration de sérum par puits en raison utilisant du sérum AB inactivé à la chaleur. Ajouter VBG + + pour obtenir un volume final de 150 pl / puits. Fermer plaque avec une feuille ELISA. </li><li> Incuber 1,5-2 h à 37 ° C tout en agitant. </li><li> Laver les globules rouges trois fois avec du PBS + BSA à 0,5% (centrifugation à 664 g pendant 2 min). </li><li> Ajouter marquées par fluorescence anti-C3 et les anticorps monoclonaux anti-C4 ou leurs fragments Fab (pour réduire l&#39;agglutination) dans du PBS + 0,5% de BSA à une concentration finale de 1 pg / ml. Voici personnalisé développé des anticorps anti-C3-Alexa Fluor 488 et anti-C4-Alexa Fluor 647 anticorps monoclonaux sont utilisés, le choix de these marqueurs fluorescents permet la détection simultanée de C3 et C4 par FACS. </li><li> Incuber 30 à 45 min à température ambiante avec agitation douce. </li><li> Laver les globules rouges 3x avec PBS + 0,5% de BSA. </li><li> Remettre en suspension les globules rouges 150 pi dans du PBS 0,5% de BSA et la pipette dans un nouveau plateau (pour se débarrasser des perles de verre avant l&#39;analyse FACS). </li><li> Effectuer une analyse FACS. Des cellules individuelles de doublets dans une dispersion de traçage FSC-A; mis la porte sur les globules rouges simples et sélectionnez le canal de détection approprié pour les signaux de fluorescence (par exemple FITC et APC). </li><li> Quantifier les résultats en utilisant l&#39;intensité médiane de fluorescence. </li></ol> Note: Il est possible d&#39;utiliser un contrôle isotypique (par exemple marqué par fluorescence anti-IL6 souris IgG1). Cependant, il est recommandé d&#39;inclure un véritable témoin négatif, par exemple par incubation de globules rouges sans sérum de patient (seulement de sérum AB) ou en l&#39;absence d&#39;une source de complément (sérum AB inactivé par la chaleur et le sérum du patient inactivé par la chaleur), und puis colorer ces avec le même anti-C3-Alexa Fluor 488 et anti-C4-Alexa Fluor 647 anticorps. On obtient ainsi un contrôle plus valide que l&#39;isotype contrôle dans le cas de globules rouges 12, bien que ces différents contrôles donnent généralement la même, résultat négatif. 3. Dosage hémolytique quantitative <ol><li> Inactiver à la chaleur la quantité requise de sérum du patient (ou une autre source d&#39;anticorps anti-RBC) par chauffage de l&#39;échantillon de 30 min à 56 ° C. </li><li> Laver les globules rouges 3x VBG - et une fois avec VBG + +. </li><li> Introduire à la pipette les éléments suivants dans une plaque à fond rond avec une perle de verre (pour un bon mélange): 35 pi 3% des globules rouges, 25 pl frais sérum AB (en complément) et 1-50% de sérum du patient. Corriger les différences de concentration dans le sérum à l&#39;aide de sérum AB inactivé par la chaleur. Ajouter VBG + + pour obtenir un volume final de 150 pl / puits. Fermer plaque avec une feuille ELISA. </li><li> Incuber à 37 ° C pendant 1,5 à 2 heures avec agitation. </li><li> Cplaque entrifuge à 664 g pendant 5 min avec une décélération réduite (par exemple de décélération pour s&#39;immobiliser dans 2-3 min). </li><li> Pipetage soigneux 90 pi surnageant dans une nouvelle plaque de microtitration. Il est important à cette étape pour éviter les bulles d&#39;air et pour éviter de prendre le long des cellules intactes. </li><li> Mesurer A414/690 dans un spectrophotomètre approprié. </li><li> Exprimer la lyse en pourcentage de la lyse des hématies d&#39;un échantillon avec de l&#39;eau pure. </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>

Les auteurs n&#39;ont rien à révéler.

Les dosages ci-dessus sont reproductibles et robuste. Ils sont faciles à réaliser et il est possible de les réaliser avec de nombreux échantillons à la fois (dans une plaque à 96 puits). Par conséquent, cette approche serait également approprié pour un système entièrement automatisé, par exemple un système de robot ELISA. Par contraste avec les techniques actuellement utilisées, ces dosages sont quantitatifs et cela aide par exemple dans l&#39;étude de l&#39;effet des inhibiteurs du comp...

Les anticorps contre les globules rouges (hématies) peut être induite par la transfusion de globules rouges exprimant un antigène qui n&#39;est pas présent sur les globules rouges de destinataire. Ces allo-anticorps peuvent provoquer de graves réactions transfusionnelles hémolytiques aiguës dues à l&#39;activation du complément sur ​​la transfusion 5 suivant. En cas d&#39;anémie hémolytique auto-immune (AHAI), les patients ont des auto-anticorps contre leurs propres globules rouges. Cela conduit à la clairance accélérée des cellules via l&#39;interaction de l&#39;IgG lié à hématies avec les récepteurs Fcy sur des phagocytes dans la rate et / ou dans les cas d&#39;auto-anticorps capables d&#39;activer le complément par l&#39;intermédiaire de récepteurs du complément dans le foie 6,7. L&#39;activation du complément fulminante résultant de l&#39;hémolyse intravasculaire est rare mais souvent fatale. Accélérée, compléter la destruction des hématies médiation induite soit par des résultats d&#39;allo-ou auto-anticorps dans l&#39;anémie aiguë et une hypoxie tissulaire donc potentiellement mortelle. Les auto-anticorps dans AHAI sont classés dans les anticorps chauds et froids, depending à la température optimale, ils se lient à des globules rouges (37 ° C ou plus bas, respectivement). Les anticorps chauds sont généralement de l&#39;isotype IgG et les anticorps IgM froides de l&#39;isotype 8,9. AIHA peut être secondaire à des troubles lymphoprolyferative par exemple, des maladies des tissus conjonctifs, les tumeurs solides, des infections ou des médicaments, mais dans 50% des cas AIHA 9 est idiopathique. Détection des gammaglobulines (p. ex. IgG ou IgM) et compléter lié à globules rouges du patient est réalisée au moyen d&#39;un Coombs direct semi-quantitative (Coombs) d&#39;essai (DAT). Dans la DAT globules rouges de patients sont incubés avec des anticorps anti-IgG ou anti-C3d. Présence de RBC agglutination démontre la présence de composants du complément attachés ou liaison de l&#39;IgG. Détection des allo-ou auto-anticorps dans le sérum du patient est réalisée par l&#39;intermédiaire du test de Coombs indirect (IAT). Dans l&#39;IAT, les globules rouges de test bromélaïne traitées sont incubées avec le sérum du patient, lavées puis incubées avec des anticorps anti-human IgG. Dans le cas où les globules rouges ont été sensibilisées avec un anticorps anti-IgG RBC présent dans le sérum du patient d&#39;agglutination va se produire. IgM anti-globules rouges mènera directement à l&#39;agglutination des globules rouges lors de l&#39;incubation de test bromélaïne traités avec le sérum du patient. RBC agglutination dans le test de Coombs direct ou dans le TAI est évaluée visuellement, soit à l&#39;oeil dans un tube à essai ou en chargeant l&#39;échantillon sur une petite colonne de Sephadex séparer les globules rouges agglutinés et simples selon la taille 1. Une autre technique fréquemment utilisée pour mesurer l&#39;activation du complément sur ​​les globules rouges est le dosage hémolytique 1, dans lequel la capacité du sérum du patient pour induire (médiée par le complément) hémolyse des hématies de bromélaïne traité 10 est évaluée. Le test est noté comme positif lorsque le surnageant après centrifugation est coloré en rouge en raison de l&#39;hémoglobine libérée et considéré comme négatif si elle reste incolore. Tant le test de Coombs et le dosage hémolytique sont semi-quantitative, depuis la plus haute serum dilution est notée dans lequel le test est toujours positif. Le test de Coombs et le dosage hémolytique sont des tests robustes qui sont couramment utilisés dans le diagnostic. Étant donné que ces tests sont semi-quantitative et en fonction de l&#39;expérience du technicien effectuant le test, ils ne sont pas adaptés à l&#39;étude des différences subtiles de l&#39;activation du complément sur les globules rouges, en fonction des besoins lors de l&#39;évaluation de l&#39;efficacité des inhibiteurs du complément. Par conséquent, nous avons développé deux essais quantitatifs pour déterminer l&#39;activation du complément par (auto)-anticorps de globules rouges, que nous décrirons dans le présent document. Tout d&#39;abord, nous avons développé un dosage pour mesurer le dépôt de fragments d&#39;activation du complément C3 et C4 sur les globules rouges (figure 1A). Dans cet essai, la bromélaïne traitée humains de type 0 globules rouges sont mis à incuber avec du sérum inactivé à la chaleur du patient (source d&#39;anticorps anti-RBC), de sérum AB frais (source de complément) et anti-C5 anticorps monoclonal (eculizumab). Au cours de cette incubation, C3 et C4 dépôt se produit si le sérum du patient contient de compléter l&#39;activation des anticorps anti-RBC. Afin d&#39;empêcher la lyse RBC par l&#39;activation du complément en aval d&#39;un anticorps monoclonal anti-C5 est ajouté blocage. Ensuite, C3 et C4 sur le dépôt des hématies est détecté par FACS en utilisant des anticorps monoclonaux marqués par fluorescence ou des fragments Fab réagissant avec C3 et C4, respectivement. Déclenchement sur simple globules rouges est important de s&#39;assurer de la fiabilité des résultats. Les avantages de cette technique sont que d&#39;un petit volume de matière du patient est nécessaire, l&#39;activation du complément à un stade précoce de la cascade est évaluée et le procédé est reproductible et quantitative. Un avantage supplémentaire de la recherche à la fois à C3 et C4 est de faire une distinction peut être faite entre l&#39;activation et la lectine voie classique (à la fois C3 et C4 dépôt) et l&#39;activation de la voie alterne (seulement de dépôt C3). L&#39;utilisation d&#39;hématies de bromélaïne traitée au lieu de globules rouges non traités augmente la sensibilité de l&#39;endire. Le second test est basé sur le dosage hémolytique actuellement utilisée (Figure 1B). Bromélaïne traitée humaines de type 0 globules rouges sont mis à incuber avec du sérum inactivé à la chaleur du patient (source d&#39;anticorps anti-RBC) et du sérum AB frais (source de complément). Si le complément est activé par des anticorps anti-RBC patients, dose-dépendante lyse RBC aura lieu, entraînant la libération de l&#39;hémoglobine. La quantité d&#39;hémoglobine libérée est quantifiée en mesurant son absorbance à 414 nm dans le surnageant après filage en bas des globules rouges intacts et fragmentés. L&#39;absorbance est en corrélation avec la quantité d&#39;hémolyse produite. Contrairement au dosage utilisé actuellement, ce protocole permet à un objectif, la valeur quantitative de l&#39;hémolyse qui est très reproductible et ne dépend pas de la personne qui interprète le dosage. Une application de ces méthodes a été décrite en 11, où l&#39;utilisation potentielle de C1-inhibiteur comme inhibiteur du complément dans l&#39;AIHA a 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

Les anticorps contre les globules rouges (hématies) peuvent conduire à l&#39;activation du complément entraîne une clairance accélérée via les récepteurs du complément dans le foie (hémolyse extravasculaire) ou conduisant à la lyse des globules rouges intravasculaire. Allo-anticorps (par exemple ABO) ou auto-anticorps contre les antigènes RBC (comme on le voit dans l&#39;anémie hémolytique auto-immune, AIHA) conduisant à l&#39;activation du complément sont potentiellement dangereux et peuvent être - surtout quand conduisant à la lyse intravasculaire - fatale 1. Actuellement, l&#39;activation du complément en raison de (auto)-anticorps sur les globules rouges est évaluée in vitro en utilisant le test de Coombs reflétant dépôt du complément sur ​​RBC ou par un dosage hémolytique non quantitative reflétant lyse RBC 1-4. Cependant, pour évaluer l&#39;efficacité des inhibiteurs du complément, il est obligatoire de disposer de techniques quantitatives. Nous décrivons ici deux de ces techniques. Tout d&#39;abord, un dosage pour détecter C3 et C4 sur le dépôt de globules rouges qui est induite par des anticorps dans le sérum du patient est préprésentés. Pour cela, on utilise une analyse par FACS avec anticorps anti-C3 ou C4 anti-marquées par fluorescence. Ensuite, une analyse quantitative hémolytique est décrite. Dans ce dosage, l&#39;hémolyse, médiée par le complément induite par le sérum du patient est mesurée usage de la fabrication de détection spectrophotométrique de l&#39;hémoglobine libérée. Ces deux essais sont très reproductible et quantitative, ce qui facilite les études de l&#39;activation du complément induite par des anticorps.

La figure 2A montre un nuage de points représentant des globules rouges. Déclenchement adapté pour les globules rouges simples peut être vu dans de FSC-W - FSC Une parcelle (P1). Habituellement, environ 95% des globules rouges entrent dans cette grille de P1 (cellules simples), mais quand un pourcentage élevé de sérum du patient est utilisé, cela peut tomber à 70-80%, en particulier lorsque des IgM anti-RBC est présent dans le sérum du patient. Les résultats repr...

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

Nous décrivons ici deux dosages pour mesurer l&#39;activation du complément induite par des anticorps contre les globules rouges. L&#39;avantage majeur sur les tests actuels est leur nature quantitative et facile à interpréter.

Les méthodes de détection quantitative d&#39;anticorps induite par l&#39;activation du complément sur les globules rouges

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 vues.  University of Amsterdam.  Nous décrivons ici deux dosages pour mesurer l'activation du complément induite par des anticorps contre les globules rouges. L'avantage majeur sur les tests actuels est leur nature quantitative et facile à interpréter. 

Vidéo: 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.

Appauvrissement des populations de cellules spécifiques par l&#39;épuisement Complément

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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Immunologie et infection

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.

L&#39;isolement des cellules de souris cavité péritonéale

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.

Modèle murin de CD40-activation des cellules 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.

Détection de Neu1 activité sialidase dans la régulation activation du récepteur Toll-like

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).

Combinaison de adhésif à base de bandes d&#39;échantillonnage et de fluorescence In situ</emHybridation> pour la détection rapide des Salmonella Sur les produits frais

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.

Protocole pour vaccins recombinants contre le SRAS RBD-base: préparation de protéines, vaccination des animaux et de détection de neutralisation

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.

Méthode de détection à haut débit pour les virus de la grippe

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.

Protégé antigènes fonctionnels globules rouges par la membrane greffage de compacts Polyglycérols hyperbranchés

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.

Les analyses qualitatives et quantitatives pour la détection et la caractérisation des antimicrobiens en protéines

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.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

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