SZ e DW receber uma subvenção irrestrita de Viropharma.

1. Preparação de tratados com bromelina Glóbulos Vermelhos <ol><li> Glóbulos vermelhos Lave 0 tipados 3x com PBS 1x (centrifugação a 664 xg por 2-5 min). </li><li> Incubar 1 volume de concentrado de eritrócitos com dois volumes de uma solução bromelina 0,5% por 10 min a 37 ° C. </li><li> Lavar as células 3x com PBS 1x. </li><li> As células podem ser armazenadas como uma solução a 3% a 4 ° C em 1 x PBS durante pelo menos uma semana. </li></ol> 2. C3 e C4 Análise Deposição sobre GVs por FACS <ol><li> Heat-inactivar a quantidade necessária de soro de um paciente (ou outra fonte de anticorpos anti-RBC) por aquecimento da amostra durante 30 min a 56 ° C. </li><li> Lavar as hemácias tratados com bromelaina três vezes em VBG - (mM veronal 5, NaCl 150 mM, gelatina a 0,05%, pH 7,4) e ressuspender-los em VBG + + (VBG - + 2 mM MgCl2 + 10 mM de CaCl 2) para dar um solução a 0,5%. </li><li> Pipetar os seguintes componentes em uma placa de fundo redondo com uma pérola de vidro (fou mistura adequada): 25 ul de 0,5% hemácias, 37,5 mL de soro fresco AB (fonte de complemento) e 37,5 mL de 200 mg / ml anti-C5. Anti-C5 é caro e pode ser difícil de obter. Pode-se considerar o uso de soro C5-/C6-/C7- ou C8-deficiente em vez de soro AB e anti-C5. </li><li> Adicionar soro por poço para uma concentração final de 0,1-33%. Corrigir as diferenças na concentração de soro por poço usando devido inactivado por calor soro AB. Adicionar VBG + + para obter um volume final de 150 ul / poço. Fechar prato com papel alumínio ELISA. </li><li> Incubar 1,5-2 h a 37 ° C enquanto se agita. </li><li> Lavar as hemácias três vezes com PBS + BSA a 0,5% (de centrifugação a 664 xg durante 2 minutos). </li><li> Adicionar marcado por fluorescência anti-C3 e os anticorpos monoclonais anti-C4 ou os seus fragmentos Fab (para reduzir a aglutinação) em PBS + BSA a 0,5% para uma concentração final de 1 ug / ml. Aqui costume desenvolveram anticorpos anti-C3-Alexa Fluor 488 e 647 de anticorpos monoclonais anti-C4-Alexa Fluor são usados, a escolha dos these marcadores fluorescentes permite a detecção simultânea de C3 e C4 por FACS. </li><li> Incubar 30-45 min à temperatura ambiente com agitação suave. </li><li> Lavar 3x com hemácias PBS + BSA a 0,5%. </li><li> Ressuspender hemácias 150 mL em PBS 0,5% BSA e pipeta-los em uma nova placa (para se livrar das pérolas de vidro antes da análise FACS). </li><li> Realizar análise FACS. Células individuais separados de parelhas em uma dispersão plotagem FSC-A; definir o portão nas hemácias individuais, selecione o canal de detecção apropriado para os sinais de fluorescência (por exemplo, FITC e APC). </li><li> Quantificar os resultados utilizando a intensidade média de fluorescência. </li></ol> Nota: É possível a utilização de um controlo do isotipo (por exemplo, marcado por fluorescência anti-IL6 IgG1 de ratinho). No entanto, recomenda-se a incluir um verdadeiro controlo negativo, por exemplo, através da incubação de eritrócitos sem soro do paciente (apenas soro AB) ou sem uma fonte de complemento (inactivado por calor soro AB e soro de um paciente inactivado pelo calor), umad em seguida corar estas com o mesmo anti-C3-Alexa Fluor 488 e anti-C4-Alexa Fluor 647 anticorpos. Isso dá um controlo mais válido do que um controlo de isotipo, no caso de células de glóbulos vermelhos 12, embora estas diferentes controlos tipicamente dar o mesmo resultado, negativo. 3. Ensaio Hemolítica Quantitative <ol><li> Heat-inactivar a quantidade necessária de soro do paciente (ou outra fonte de anticorpos anti-RBC) por aquecimento da amostra de 30 min a 56 ° C. </li><li> Lavar 3x com hemácias VBG - e uma vez com VBG + +. </li><li> Pipetar os seguintes componentes em uma placa de fundo redondo com uma pérola de vidro (para mistura adequada): 35 mL de 3% hemácias, 25 mL fresco soro AB (fonte de complemento) e 1-50% de soro do paciente. Corrigir as diferenças na concentração de soro inactivado por calor que utilizam soro AB. Adicionar VBG + + para obter um volume final de 150 ul / poço. Fechar prato com papel alumínio ELISA. </li><li> Incubar a 37 ° C durante 1,5-2 h, com agitação. </li><li> Cplaca entrifuge a 664 xg por 5 min com desaceleração reduzida (por exemplo, a desaceleração ponto final em 2-3 min). </li><li> Pipetar cuidadosamente 90 mL de sobrenadante em uma nova placa de microtitulação. É importante nesta etapa para evitar bolhas de ar e impedir a levar células intactas. </li><li> Meça A414/690 em espectrofotômetro adequado. </li><li> Exprime-se o lise como uma percentagem da lise de uma amostra GVs com água pura. </li></ol>

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

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Os ensaios acima são reprodutíveis e robusto. Eles são fáceis de realizar e é possível realizá-las com várias amostras ao mesmo tempo (em uma placa de 96 poços). Assim, esta abordagem também seria adequado para um sistema totalmente automatizado, por exemplo, um sistema robotizado de ELISA. Em contraste com as técnicas actualmente utilizadas, estes ensaios são quantitativos e isso irá ajudar, por exemplo, no estudo do efeito de inibidores de complemento. Além disso, a interpretação é o...

Os anticorpos contra as células vermelhas do sangue (RBC) pode ser induzida por transfusão de glóbulos vermelhos que expressam um antigénio, que não está presente em eritrócitos de destinatários. Esses anticorpos podem causar graves alo reações transfusionais hemolíticas agudas devido à ativação do complemento no seguinte transfusão 5. Na anemia hemolítica auto-imune (AHAI), os pacientes têm auto-anticorpos contra suas próprias hemácias. Isto leva a folga acelerada das células através da interacção de IgG ligada a glóbulos vermelhos com receptores Fcy em fagócitos do baço e / ou no caso de auto-anticorpos capazes de activar o complemento por meio de receptores de complemento no fígado 6,7. Ativação do complemento fulminante resultando em hemólise intravascular é raro, mas muitas vezes fatal. Acelerado, complementam destruição de hemácias mediada induzida por qualquer auto-anticorpos ou alo resulta em anemia aguda e hipóxia tecidual, portanto, potencialmente fatal. Os auto-anticorpos em AHAI são classificadas em anticorpos quentes e frios, depending na temperatura ideal para se ligar glóbulos vermelhos (37 ° C ou inferiores, respectivamente). Os anticorpos são geralmente quentes de isotipo IgG e os anticorpos frios do isotipo IgM 8,9. AHAI pode ser derivado por exemplo, de lymphoprolyferative distúrbios, doenças dos tecidos conjuntivos, os tumores sólidos, infecções ou drogas, mas em 50% dos casos é AHAI idiopática 9. Detecção de gamaglobulina (por exemplo. IgG ou IgM) e complementar ligada a glóbulos vermelhos do doente é realizado por meio de um teste da antiglobulina semiquantitativa directa (Coombs) (DAT). No DAT GVs do paciente são incubadas com anti-IgG ou anti-C3d. Ocorrência de RBC aglutinação demonstra a presença de componentes do complemento em anexo ou ligação IgG. A detecção de anticorpos no soro do paciente-alo ou é realizada por meio do teste da antiglobulina indirecto (TAI). No IAT, hemácias de ensaio tratados com bromelaina são incubadas com o soro de um paciente, lavadas e depois incubadas com anti-human IgG. No caso de eritrócitos foram sensibilizados com anticorpos anti-eritrócitos de IgG presente no soro do paciente a aglutinação ocorrerá. Anticorpos IgM para hemácias levará diretamente para a aglutinação de hemácias na incubação de teste tratadas com bromelina com soro do paciente. RBC aglutinação no teste de Coombs direto ou no IAT é visualmente avaliadas por meio de olho em um tubo de ensaio ou por carregar a amostra em uma pequena coluna de Sephadex separa os glóbulos vermelhos aglutinados e individuais por tamanho 1. Uma outra técnica frequentemente utilizada para medir a activação do complemento em eritrócitos é o ensaio hemolítico 1, em que a capacidade do soro de um paciente para induzir (mediada pelo complemento) hemólise de eritrócitos tratados com bromelaina 10 é avaliada. O teste é conhecido como positivo quando o sobrenadante após a centrifugação está manchado de vermelho devido à hemoglobina liberada e considerado negativo se ele permanece incolor. Tanto o teste da antiglobulina e o ensaio hemolítico são semi-quantitativo, uma vez que a maior serum diluição é denotado na qual o teste ainda é positivo. O teste da antiglobulina e o ensaio hemolítico são ensaios robustos que são rotineiramente utilizados em diagnósticos. Dado que estes ensaios são semi-quantitativo e depende da experiência do técnico da realização do ensaio, não são apropriados para estudar as diferenças subtis da activação do complemento em eritrócitos, conforme necessário ao avaliar a eficácia de inibidores de complemento. Por isso, desenvolvemos dois ensaios quantitativos para determinar a ativação do complemento por anticorpos (auto-) de hemácias, que vamos descrever neste artigo. Em primeiro lugar, foi desenvolvido um ensaio para medir a deposição de fragmentos de activação do complemento C3 e C4 no RBC (Figura 1A). Neste ensaio, os humanos tratados com bromelaina tipo-0 GVs são incubadas com soro inactivado por calor paciente (fonte de anticorpos anti-RBC), soro AB fresco (fonte de complemento) e anticorpo monoclonal anti-C5 (eculizumab). Durante este incubation, a deposição de C3 e C4 ocorrerá se o soro do paciente contém complemento activando anticorpos anti-RBC. A fim de evitar lise por activação do complemento a jusante, é adicionado um anticorpo monoclonal anti-C5 de bloqueio. Em seguida, a deposição de C3 e C4 no RBC é detectada por FACS utilizando anticorpos monoclonais marcados com fluorescência ou fragmentos Fab que reagem com C3 e C4, respectivamente. Gating em único hemácias é importante para garantir a confiabilidade dos resultados. As vantagens desta técnica são as de que um pequeno volume de material paciente é necessária, a activação do complemento na fase inicial da cascata é avaliada e o método é reproduzível e quantitativo. Uma vantagem adicional de olhar para ambos C3 e C4 é que pode ser feita uma distinção entre a ativação clássica e lectina caminho (ambos C3 e C4 deposição) ea ativação da via alternativa (apenas C3 deposição). A utilização de eritrócitos tratados com bromelaina, em vez de RBC não tratada aumenta a sensibilidade do quantodizer. O segundo ensaio é baseado no ensaio hemolítico actualmente utilizado (Figura 1B). Humanos tratados com bromelina do tipo 0 hemácias são incubadas com soro inativado pelo calor do paciente (fonte de anticorpos anti-RBC) e soro AB fresca (fonte de complemento). Se o complemento é ativado por anticorpos anti-RBC paciente, dependente da dose de lise RBC irá ocorrer, levando à liberação de hemoglobina. A quantidade de hemoglobina libertada é quantificada através da medição da sua absorvância a 414 nm no sobrenadante depois de rodar para baixo as hemácias intactos e fragmentados. A absorvência está correlacionada com a quantidade de hemólise ocorreu. Em contraste com o ensaio actualmente utilizada, este protocolo permite a um objectivo, o valor quantitativo de hemólise que é muito reprodutível e não dependente da pessoa interpreta o ensaio. Uma aplicação destes métodos tem sido descrita em 11, onde o uso potencial de C1-inibidor como inibidor do complemento na AHAI foi 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

Os anticorpos contra os glóbulos vermelhos (hemácias) pode levar a ativação do complemento, resultando em um procedimento acelerado através de receptores do complemento no fígado (hemólise extravascular) ou levando a lise intravascular de hemácias. Aloanticorpos (por exemplo, ABO) ou auto-anticorpos contra os antigénios de RBC (como visto na anemia hemolítica auto-imune, AIHA) que conduzem à activação do complemento são potencialmente prejudiciais e podem ser - especialmente quando conduzindo à lise intravascular - fatal 1. Atualmente, a ativação do complemento devido a (auto)-anticorpos em hemácias é avaliada in vitro através do teste de Coombs refletindo deposição de complemento na RBC ou por um ensaio hemolítico não quantitativa refletindo de lise dos 1-4. No entanto, para avaliar a eficácia de inibidores de complemento, é obrigatória a existência de técnicas quantitativas. Aqui nós descrevemos dois tais técnicas. Em primeiro lugar, um ensaio para detectar a deposição de C3 e C4 em células vermelhas do sangue que é induzido por anticorpos no soro de um paciente é préapresentadas. Por isso, a análise de FACS é usado com os anticorpos anti-C3 ou anti-C4 marcados com fluorescência. Em seguida, um ensaio hemolítico quantitativa é descrito. Hemólise Neste ensaio, mediada pelo complemento induzida por soro do doente é medido fazendo uso de detecção espectrofotométrica da hemoglobina libertada. Ambos os ensaios são muito reprodutíveis e quantitativa, facilitando estudos de activação de complemento induzida pela anticorpo.

Figura 2A mostra um gráfico representativo de dispersão para hemácias. Gating adequado para eritrócitos individuais pode ser vista em de FSC-W - FSC-A trama (P1). Normalmente, cerca de 95% dos GVs são abrangidas por esta porta P1 (células isoladas), mas quando uma elevada percentagem de soro do paciente é usado, este pode cair para 70-80%, especialmente quando o anti-RBC IgM está presente no soro do paciente. Os resultados representativos para o efeito de AHAI soro d...

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

Aqui nós descrevemos dois ensaios para medir a ativação do complemento induzida por anticorpos contra células vermelhas do sangue. A vantagem principal sobre os ensaios actuais é a sua natureza quantitativa e fácil de interpretar.

Métodos para a detecção quantitativa de anticorpos induzida pela ativação do complemento no Glóbulos Vermelhos

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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JoVE Journal

Immunology and Infection

30.4K visualizações.  University of Amsterdam.  Aqui nós descrevemos dois ensaios para medir a ativação do complemento induzida por anticorpos contra células vermelhas do sangue. A vantagem principal sobre os ensaios actuais é a sua natureza quantitativa e fácil de interpretar. 

Vídeo: 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.

Esgotamento das populações de células específicas de Esgotamento Complemento

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

Imunologia e Infecção

Isolation of Mouse Peritoneal Cavity Cells

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

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

Isolamento de células de camundongo Peritoneal Cavity

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.

Modelo murino de CD40-ativação de células 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.

Detecção de Atividade Neu1 sialidase em Regulação Toll-like receptor de ativação

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).

Combinação de adesivos de fita baseada em amostragem e de fluorescência In situ Hibridação para detecção rápida de 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 ...

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.

Protocolo para vacinas recombinantes RBD baseado SARS: Preparação de proteínas, Vacinação Animal e Detecção de Neutralização

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.

Alta capacidade método de detecção de vírus Influenza

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

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

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

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

Antígenos funcionais protegidos glóbulos vermelhos pela membrana enxerto de compactos Poligliceróis hiper

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.

Qualitativos e quantitativos Os ensaios para a detecção e caracterização de antimicrobianos Proteína

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 aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

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