SZ und DW erhalten eine zweckgebundene Zuwendung von Viropharma.

1. Herstellung von Bromelain-behandelten roten Blutzellen <ol><li> Waschen 0 typisierte roten Blutkörperchen 3x mit 1x PBS (Zentrifugation bei 664 g für 2-5 min). </li><li> Inkubieren 1 Volumen Erythrozytenkonzentrat mit zwei Volumina einer 0,5% Bromelain Lösung für 10 min bei 37 ° C. </li><li> Waschen Sie die Zellen 3x mit 1x PBS. </li><li> Die Zellen können als eine 3% ige Lösung bei 4 ° C in 1 × PBS für mindestens eine Woche gelagert werden. </li></ol> 2. C3 und C4 Deposition Analyse auf RBCs durch FACS <ol><li> Wärme-Inaktivierung die erforderliche Menge an Patientenserum (oder andere anti-RBC Antikörper Quelle) durch Erhitzen der Probe für 30 min bei 56 ° C. </li><li> Dreimal waschen Bromelain behandelten Erythrozyten in VBG - (5 mM Veronal, 150 mM NaCl, 0,05% Gelatine, pH 7,4) und resuspendieren sie in VBG + + (VBG - + 2 mM MgCl 2 + 10 mM CaCl 2), um ein zu geben 0,5% igen Lösung. </li><li> Pipettieren Sie die folgenden Komponenten in einem Rundbodenplatte mit einer Glasperle (foder richtige Mischung): 25 ul 0,5% Erythrozyten, 37,5 ul frisch AB-Serum (Komplement-Quelle) und 37,5 ul 200 ug / ml anti-C5. Anti-C5 ist teuer und vielleicht schwer zu bekommen sein. Man könnte überlegen, C5-/C6-/C7- oder C8-defizienten Serum anstelle von AB-Serum und Anti-C5. </li><li> Hinzufügen Patientenserum pro Vertiefung bis zu einer Endkonzentration von 0,1 bis 33%. Richtige für Unterschiede in der Serumkonzentration pro Vertiefung durch Verwendung von hitzeinaktiviertem AB-Serum. In VBG + +, um eine endgültige Volumen von 150 ul / gut. Schließen ELISA-Platte mit Folie. </li><li> Inkubieren 1,5-2 h bei 37 ° C unter Schütteln. </li><li> Die Erythrozyten dreimal mit PBS + 0,5% BSA (Zentrifugation bei 664 × g für 2 min) waschen. </li><li> Hinzufügen fluoreszenzmarkierten anti-C3 und C4 monoklonalen anti-Antikörper oder deren Fab-Fragmente in PBS + 0,5% BSA bis zu einer Endkonzentration von 1 ug / ml (um die Agglutination zu verringern). Hier speziell entwickelte Anti-C3-Alexa Fluor 488 und Anti-C4-Alexa Fluor 647 monoklonale Antikörper verwendet werden, die Wahl der these fluoreszierenden Markierungen erlaubt die gleichzeitige Detektion von C3 und C4 durch FACS. </li><li> Inkubiere 30-45 Minuten bei Raumtemperatur unter leichtem Schütteln inkubiert. </li><li> Wash Erythrozyten 3x mit PBS + 0,5% BSA. </li><li> Resuspendieren RBC 150 ul in PBS + 0,5% BSA und pipettieren sie in eine neue Platte (auf der Glasperlen loswerden, bevor FACS Analyse). </li><li> FACS-Analyse durchführen. Separate Einzelzellen von Dubletten in einem Streu Plotten FSC-A; setzen das Tor zu den einzelnen Erythrozyten, wählen Sie die entsprechende Detektionskanal für die Fluoreszenz-Signale (z. B. FITC und APC). </li><li> Quantifizieren Ergebnisse mit dem Median der Fluoreszenzintensität. </li></ol> Hinweis: Es ist möglich, eine Isotyp-Kontrolle (zB Fluoreszenz-markierten anti-IL-6-Maus-IgG1) zu verwenden. Es wird jedoch empfohlen, um eine echte Negativkontrolle zB durch Inkubation von Erythrozyten ohne Patientenserum (nur AB-Serum) oder ohne Ergänzung Quelle (hitzeinaktiviertem AB-Serum und Hitze-inaktiviertes Patientenserum) gehören, eind dann färben diese mit der gleichen anti-C3-Alexa Fluor 488 und Anti-C4-Alexa Fluor 647-Antikörper. Dies ergibt eine weitere gültige Steuer als eine Isotyp-Kontrolle im Falle der roten Blutzellen 12, wobei diese verschiedenen Steuerungen geben typischerweise die gleichen, negativen Ergebnis. 3. Quantitative hämolytisch-Assay <ol><li> Wärme-Inaktivierung die erforderliche Menge von Patientenseren (oder andere anti-RBC Antikörper Quelle) durch Erhitzen der Probe 30 min bei 56 ° C. </li><li> Wash Erythrozyten 3x VBG - und einmal mit VBG + +. </li><li> Pipettieren Sie die folgenden Komponenten in einem Rundbodenplatte mit einer Glasperle (für die richtige Durchmischung): 35 ul 3% Erythrozyten, 25 ul frisch AB-Serum (Komplement-Quelle) und 1-50% Patientenserum. Richtige für Unterschiede im Serum-Konzentration mit hitzeinaktiviertem AB-Serum. In VBG + +, um eine endgültige Volumen von 150 ul / gut. Schließen ELISA-Platte mit Folie. </li><li> Bei 37 ° C für 1,5 bis 2 h mit Schütteln. </li><li> Centrifuge Platte bei 664 × g für 5 min mit reduzierten Verzögerung (z. B. Verzögerung zu Punkt in 2-3 min). </li><li> Vorsichtig pipettieren, 90 ul Überstand in eine neue Mikrotiterplatte. Es ist wichtig, in diesem Schritt, um Luftblasen zu verhindern und um zu verhindern, entlang intakten Zellen zu übernehmen. </li><li> Messen Sie A414/690 in einem geeigneten Spektrophotometer. </li><li> Ausdruck der Lyse in Prozent Lyse einer Probe RBCs mit reinem Wasser. </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>

Die Autoren haben nichts zu offenbaren.

Die obigen Tests sind reproduzierbar und stabil. Sie sind einfach durchzuführen und es ist möglich, sie mit vielen Proben gleichzeitig (in einer 96-well-Platte) durchzuführen. Daher würde dieser Ansatz auch für ein vollautomatisches System, z. B. eine ELISA-Robotersystem. Im Gegensatz zu den gegenwärtig verwendeten Techniken sind diese Assays quantitativ und dies wird in der Studie über die Wirkung von Komplement-Inhibitoren helfen zB. Außerdem ist die objektive Interpretation, welche eine Verb...

Antikörper gegen rote Blutkörperchen (Erythrozyten) durch Transfusion von roten Blutkörperchen, die ein Antigen, das nicht auf Empfänger RBCs vorhanden ist exprimieren, induziert werden. Diese Allo-Antikörper können schwere akute hämolytische Transfusionsreaktionen aufgrund der Aktivierung auf die nach der Transfusion 5 ergänzen verursachen. In Auto-Immun-hämolytische Anämie (AIHA) haben die Patienten Auto-Antikörper gegen ihre eigenen roten Blutkörperchen. Dies führt zu einer beschleunigten Clearance der Zellen durch die Interaktion von IgG mit Fc &amp;ggr; RBC-Rezeptoren auf Phagocyten in der Milz und / oder im Falle von Autoantikörpern in der Lage, mittels Komplement-Komplement-Rezeptoren in der Leber 6,7 aktivieren gebunden. Fulminante Komplement-Aktivierung intravaskulären Hämolyse was ist selten, aber oft tödlich. Beschleunigte ergänzen vermittelten RBC Zerstörung entweder durch Allo-oder Autoantikörpern führt zu akuter Anämie und damit potenziell tödlichen Hypoxie induziert. Die Autoantikörper in AIHA sind in warmen und kalten Antikörper eingestuft, dependinG auf der optimalen Temperatur sie Erythrozyten (37 ° C oder geringer) zu binden. Die warmen Antikörper sind in der Regel von IgG-Isotyp und die kalten Antikörper vom IgM-Isotyp 8,9. AIHA kann sekundär zB lymphoprolyferative Erkrankungen, Bindegewebserkrankungen, soliden Tumoren, Infektionen oder Medikamente sein, aber in 50% der Fälle ist idiopathischen AIHA 9. Detektion Gammaglobuline (zB. IgG oder IgM) und Komplement gebunden Patienten RBCs mittels einer semiquantitativen direkten Antiglobulin (Coombs)-Test (DAT) durchgeführt. In dem DAT-Patienten RBCs mit Anti-IgG-oder Anti-C3d inkubiert. Das Auftreten von RBC Agglutination zeigt die Anwesenheit von Komplement-Komponenten befestigt oder IgG-Bindung. Nachweis von Allo-oder Autoantikörpern im Serum des Patienten wird mittels der indirekten Antiglobulintest (IAT) durchgeführt. In der IAT werden Bromelain behandelten Test RBCs mit Patientenserum inkubiert, gewaschen und dann mit anti-h inkubiertuman IgG. Im Falle haben RBCs mit Anti-RBC-IgG, die in dem Patientenserum Agglutination auftritt sensibilisiert. IgM-Antikörper gegen Erythrozyten wird direkt an Agglutination nach der Inkubation von Bromelain-behandelten Test Erythrozyten mit Patientenserum führen. RBC Agglutination in der direkten Coombs-Test oder im IAT wird optisch entweder mit dem Auge in einem Reagenzglas oder durch das Laden der Probe auf einer kleinen Sephadexsäule Trennung agglutinierten Erythrozyten-und Einzel nach Größe 1 bewertet. Ein anderes häufig verwendetes Verfahren zur Komplementaktivierung auf RBCs messen, ist der hämolytischen Assay 1, bei dem die Kapazität von Patientenserum (Komplement-vermittelte) Hämolyse von Bromelain behandelten RBC 10 zu induzieren, beurteilt. Der Test wird als positiv festgestellt, wenn der Überstand nach dem Zentrifugieren wird rot wegen freigesetzte Hämoglobin gefärbt und als negativ zu sein, wenn es farblos bleibt. Sowohl das Coombs-Test und der Test sind hämolytische semiquantitative, da die höchste serum Verdünnung bezeichnet, in dem der Test ist immer noch positiv. Der Coombs-Test und dem hämolytisch-Assay sind robust Tests, die routinemäßig in der Diagnostik eingesetzt werden. Da diese Tests sind semiquantitative und abhängig von der Erfahrung des Technikers Durchführung des Assays sie geeignet ist, subtile Unterschiede der Komplementaktivierung auf Erythrozyten zu untersuchen sind nicht, wie erforderlich, wenn die Bewertung der Wirksamkeit von Komplement-Inhibitoren. Daher entwickelten wir zwei quantitative Tests, um die Komplement-Aktivierung von (Auto-) Antikörper gegen Erythrozyten, die wir in diesem Papier beschreiben, zu bestimmen. Zuerst wird ein Test, um die Ablagerung von Komplement-Aktivierung Fragmente C3 und C4 auf RBCs (1A) messen wir entwickelt. In diesem Test werden menschliche Bromelain behandelten Typ 0 RBCs mit hitzeinaktiviertem Patientenserum (anti-RBC Antikörper Quelle), frisch AB-Serum (Komplement-Quelle) und Anti-C5-Antikörper (Eculizumab) inkubiert. Während dieser inc.ubation wird C3 und C4 Abscheidung auftreten, wenn das Patientenserum enthält ergänzen Aktivierung anti-RBC Antikörper. Um RBC Lyse durch nachgeschaltete Komplementaktivierung verhindern eine Sperr Anti-C5-monoklonaler Antikörper wird hinzugefügt. Als nächstes wird C3 und C4 Abscheidung auf der RBCs durch FACS unter Verwendung von Fluoreszenz-markierten monoklonalen Antikörpern oder Fab-Fragmenten Umsetzung mit C3 und C4, die jeweils detektiert. Gating auf einzelne Erythrozyten ist wichtig, um die Zuverlässigkeit der Ergebnisse zu gewährleisten. Vorteile dieses Verfahrens beinhalten, dass ein kleines Volumen des Patientenmaterials erforderlich ist, Komplementaktivierung in einer frühen Stufe der Kaskade bewertet wird und das Verfahren ist reproduzierbar und quantitativ. Ein weiterer Vorteil der Blick auf beide C3 und C4 ist, dass eine Unterscheidung zwischen der klassischen und Lektin-Weg-Aktivierung (sowohl C3-und C4-Deposition) und den alternativen Weg der Aktivierung (nur C3 Deposition) hergestellt werden. Die Verwendung von Bromelain behandelten Erythrozyten statt unbehandelten roten Blutkörperchen erhöht die Empfindlichkeit der alszu sagen. Der zweite Test basiert auf der gegenwärtig verwendeten hämolytischen Assay (Fig. 1B) auf. Menschen Bromelain-behandelten Typ-0-Erythrozyten werden mit Hitze-inaktiviertem Patientenserum (anti-RBC Antikörper-Quelle) und frischem AB-Serum (Komplement-Quelle) inkubiert. Wenn Komplement von Patienten anti-RBC-Antikörper aktiviert, wird eine dosisabhängige RBC-Lyse auftreten, was zu der Hämoglobin freizusetzen. Die Menge an freigesetztem Hämoglobin wird durch Messen seiner Absorption bei 414 nm im Überstand nach Abzentrifugieren der intakten und fragmentierten Erythrozyten quantifiziert. Die Extinktion korreliert mit der Menge der Hämolyse aufgetreten. Im Gegensatz zu den derzeit verwendeten Assay erlaubt eine objektive, quantitative Wert der Hämolyse, die sehr gut reproduzierbar und unabhängig von der Person, die Interpretation des Assays ist das Protokoll. Eine Anwendung dieser Methoden ist in 11, wobei die mögliche Verwendung von C1-Inhibitor als Komplement-Inhibitor in AIHA war s beschriebentudied.

<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

Antikörper gegen rote Blutkörperchen (Erythrozyten) kann dazu führen, Aktivierung, was zu einer beschleunigten Genehmigungs über Komplement-Rezeptoren in der Leber (extravaskulären Hämolyse) oder die zu intravaskulären Lyse von Erythrozyten zu ergänzen. Alloantikörper (zB ABO) oder Autoantikörper gegen RBC-Antigene (wie in der Autoimmun-hämolytische Anämie, AIHA zu sehen), die zur Aktivierung ergänzen sind potenziell schädlich und kann - vor allem, wenn die zu intravaskulären Lyse - tödlichen ein. Derzeit Komplement-Aktivierung durch (auto)-Antikörpern auf Erythrozyten in vitro mit Hilfe der Coombs-Test reflektierende Ergänzung Abscheidung auf RBC oder durch eine nicht-quantitative Assay hämolytische reflektieren RBC Lyse 1-4 bewertet. Um jedoch die Wirksamkeit von Inhibitoren Ergänzung zu bewerten, ist es zwingend erforderlich, um quantitative Techniken haben. Hier beschreiben wir zwei solche Techniken. Zuerst wird ein Test zur C3 und C4 Abscheidung auf rote Blutzellen, die von Antikörpern in Patientenserum induziert wird erkennen vorgepräsentierte. Hierzu ist die FACS-Analyse mit Fluoreszenz-markierten anti-C3-oder Anti-C4 Antikörper verwendet. Als nächstes wird eine quantitative hämolytischen Assay beschrieben. In diesem Assay Komplement-vermittelte Hämolyse von Patientenserum induziert ist die Nutzung der spektrophotometrischen Nachweis des freigesetzten Hämoglobins gemessen. Diese beiden Tests sind sehr reproduzierbare und quantitative erleichtert Untersuchungen der Antikörper-induzierten Komplementaktivierung.

2A zeigt eine repräsentative Streudiagramm für Erythrozyten. FSC-A-Grundstück (P1) - Geeignet für Einzel Gating Erythrozyten in de FSC-W zu sehen. In der Regel rund 95% der roten Blutkörperchen, gehören zu dieser P1-Gate (Einzelzellen), aber wenn ein hoher Prozentsatz der Patientenserum verwendet wird, kann diese auf 70-80% sinken, vor allem, wenn anti-RBC IgM im Serum des Patienten vorhanden ist. Repräsentative Ergebnisse für die Wirkung von Patientenserum auf AIHA ...

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

Hier beschreiben wir zwei Assays zur Messung von Komplement-Aktivierung durch Antikörper gegen rote Blutzellen induziert. Der große Vorteil gegenüber den aktuellen Untersuchungen ist die quantitative und einfach zu interpretieren Natur.

Methoden für die quantitative Bestimmung von Antikörper-induzierte Komplementaktivierung auf Red Blood Cells

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

methods-for-quantitative-detection-antibody-induced-complement

Research

JoVE Journal

Immunology and Infection

30.4K Aufrufe.  University of Amsterdam.  Hier beschreiben wir zwei Assays zur Messung von Komplement-Aktivierung durch Antikörper gegen rote Blutzellen induziert. Der große Vorteil gegenüber den aktuellen Untersuchungen ist die quantitative und einfach zu interpretieren Natur. 

Serie: 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.

Depletion spezifischer Zellpopulationen durch Ergänzung Depletion

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

Forschung

Immunologie und Infektion

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.

Isolierung von Maus Bauchhöhle 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 ...

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.

Mausmodell der CD40-Aktivierung von B-Zellen

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.

Erkennung von Neu1 Sialidase-Aktivität bei der Regulierung der Toll-like-Rezeptor-Aktivierung

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).

Kombination von Klebstoff-tape-basierten Sampling und Fluoreszenz In situ Hybridisierung zum schnellen Nachweis von Salmonellen 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 ...

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.

Protokoll für die rekombinante RBD-basierte Impfstoffe SARS: Protein Preparation, Animal Impfung und Neutralisation Erkennung

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 Nachweisverfahren für 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, ...

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.

Antigene geschützten funktionellen Red Blood Cells durch die Membran Pfropfung von Compact hyperverzweigten Polyglycerinen

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.

Qualitative und quantitative Assays zur Detektion und Charakterisierung von Protein Antimikrobielle

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.

Dextran erhöht die Effizienz der Lentivirale Transduktion von murinen und menschliche primäre NK-Zellen

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