Gli autori desiderano ringraziare per i finanziamenti dal NIH 3R01AI080289-02S1, e un Harvard University Center for AIDS Research Scholar Fellowship in NIH / NIAID 2P30AI060354-07.

1. Cellule cultura fagocitiche <ol><li> Cultura cellule THP-1 10 in RPMI 1640 supplementato con 10% di siero fetale bovino come una sospensione stazionario in fiasche T. Densità cellulare deve essere mantenuta al di sotto 0.5x10 6 / ml al fine di mantenere livelli coerenti di espressione FcγR e performance test. </li></ol> 2. Preparare Antigen biotinilato <ol><li> Calcolare la quantità di solfo-NHS reagente kit LC biotina necessario biotinylate l&#39;antigene bersaglio di interesse secondo le istruzioni del produttore. </li><li> Sciogliere il reagente biotinilazione in acqua e aggiungere immediatamente l&#39;importo calcolato per l&#39;antigene in un buffer che non contiene ammine primarie. Lasciare che la reazione di procedere per 1 ora a temperatura ambiente, mescolando di tanto in tanto. </li><li> Rimuovere i residui, biotina coniugata con uno scambio di buffer in un reparto di centrifuga concentrazione Amicon di un taglio appropriato peso molecolare per mantenere la Targeantigene t. Aggiungere campione alla camera superiore dell&#39;unità concentrazione e aggiungere PBS per portare il volume totale di 15 ml linea di riempimento. Centrifugare a 4000 xg per portare il volume fino a circa 1,5 ml. Ripetendo questo processo due volte elimina il 99% della biotina gratuiti che assicurano la massima rivestimento di antigene di perline. </li></ol> 3. Preparare Perline Antigen saturi <ol><li> Lavare 100 l di 1 millimetro neutravidin perline fluorescenti due volte in 1 ml 0,1% PBS-BSA dopo riducendo la velocità in una microcentrifuga ad alta velocità. Risospendere lavato perle in 100 l di PBS-BSA, e aliquote in 10 tubi. </li><li> Per determinare il rivestimento in condizioni antigene che saturano le perline, unire 10 ul della sospensione lavato tallone con diverse concentrazioni di antigene biotinilato in duplice passi. Incubare per una notte a 4 ° C in una provetta su un rotatore. </li></ol> NOTA: saturazione di perline devono essere determinati sperimentalmente. Questo può essere realizzatoidentificando bead-coating condizioni che producono la fagocitosi massima quando perline vengono successivamente opsonizzati con un anticorpo monoclonale di controllo. <ol start="3"><li> Rimuovere l&#39;antigene non legato da un lavaggio con 1 ml di PBS-BSA e centrifuga ad alta velocità fino perle sono pellet. Rimuovere PBS-BSA e ripetere. </li><li> Risospendere antigene perle rivestite in un volume finale di 1 ml in PBS-BSA. Perle possono essere conservati per un massimo di una settimana a 4 ° C prima dell&#39;uso. </li></ol> 4. Preparare i campioni anticorpo <ol><li> Campioni di anticorpi clinici può essere purificato dal plasma utilizzando un gel kit Melone IgG purificazione secondo le istruzioni del produttore. IgG purificate possono essere conservati a 4 ° C fino al momento dell&#39;utilizzo. </li></ol> NOTA: le opportune precauzioni per quanto riguarda la manipolazione di campioni umani devono sempre stata presa. <ol start="2"><li> Determinare la concentrazione di anticorpi purificati mediante assorbimento a A280, e diluire i campioni di 1 mg / ml in PBS. </li><li> Preparare postulareive e negativo anticorpi monoclonali di controllo per diluizione di 1 mg / ml in PBS. </li></ol> 5. Placcatura l&#39;esperimento <ol><li> Risospendere il lavato, soluzione satura di antigene tallone preparato in precedenza con il vortex, e trasferire 10 microlitri in ogni pozzetto di un fondo rotondo 96 pozzetti. Bisogna fare attenzione ad agitare continuamente la sospensione tallone per assicurare un numero uguale di perline fluorescenti sono aggiunti a ciascun pozzetto. </li><li> Aggiungi concentrazioni variabili degli anticorpi di interesse in ogni pozzetto, creando una curva dose-risposta per ogni anticorpo. Concentrazioni ottimali variano tra i campioni a seconda del titolo di anticorpi presenti, ma una gamma di 0,01-100 mcg / ml concentrazione finale fornirà una buona copertura e permettere di identificare l&#39;intervallo di concentrazione di interesse. Assicurarsi che gli anticorpi sono aggiunti in quantità non superiori a 20 l. </li><li> Perle di incubare e campioni di anticorpi per 2 ore a 37 ° C per consentire al anticorpi opsonize le perline. &lt;/ Li&gt; <li> Preparare una sospensione di cellule THP-1 a 2,5 x 10 5 cellule / ml e aggiungere 200 ml di questa sospensione in ogni pozzetto, per un totale di 5 x 10 4 cellule THP-1 in ciascun pozzetto. </li><li> Incubare per una notte a 37 ° C, 5% di CO 2, in un incubatore stazionario, le cellule permettendo e perline di pellet per gravità. </li></ol> NOTA: segnale al rumore può essere migliorata attraverso la determinazione del tallone ottimale: anticorpo: THP-1 rapporti di cella per una determinata sorgente antigeni e anticorpi. 6. Analisi in citometria a flusso <ol><li> Togliere 100 l di surnatante da ogni pozzetto facendo attenzione a non disturbare il pellet. Surnatante può essere salvato se determinazioni secrezione di citochine o altre analisi sono volute. </li><li> Per il fissaggio, aggiungere 100 ml di paraformaldeide 4% in ogni pozzetto e pipetta per risospendere e mescolare cellule. </li><li> Analisi dei flussi ad alta velocità citometria può essere eseguito utilizzando un BD LSR II dotato di un lettore di piastre HTS.</li><li> Programma software per mixare ogni ben tre volte (100 volumi mix microlitri) e analizzare 30 microlitri, o almeno 2000 eventi cellula, di ogni campione. </li><li> I dati raccolti possono essere analizzati in FlowJo o software equivalente. Metriche utili sono la percentuale di + tallone, o cellule fluorescenti, che fornisce una misura del numero dei fagociti presenti, così come l&#39;intensità di fluorescenza delle cellule fagocitarie, che fornisce una misura del numero di perline fagocitati. Moltiplicando questi valori genera un sistema integrato di media intensità fluorescente, o iMFI. </li><li> Il numero medio di perline fagocitati da ogni cellula fagocitica può essere calcolato dividendo il iMFI dalla fluorescenza media di 1 perlina. </li></ol> 7. Rappresentante Risultati Ci dovrebbe essere una chiara differenziazione dei campioni di anticorpi da parte di soggetti affetti e non. Figura 1A presenta gli istogrammi FACS di un campione di anticorpi da HIV negativozione (traccia nera) e un soggetto sieropositivo (traccia grigia), e dimostra la fagocitosi aumentato guidato dalla presenza di antigene-specifici anticorpi. Sensibilità ottimale del test dipende dalla saturazione delle perle con l&#39;antigene biotinilato. Figura 1B presenta la fagocitosi osservata quando perle rivestite con differenti quantità di antigene sono stati opsonizzati con 3 anticorpi monoclonali di controllo (tra cui un non-legame degli anticorpi, triangoli), e stabilisce 2μg antigene / mL come perle di una concentrazione di saturazione per questo antigene. Una curva dose-risposta è illustrato nella figura 2, e dimostra la capacità differenziale di campioni anticorpo soggetti a indurre la fagocitosi su una gamma di anticorpi concentrazione di 0,05-5 mg / ml. Questo fagocitosi differenziale può essere guidata da due differenze di titolo o di proprietà del dominio Fc come sottoclassi di IgG e lo stato di glicosilazione. Quando cellule THP-1 sono iMaged al microscopio a fluorescenza, non c&#39;è chiara evidenza di fagocitosi tallone. La figura 3 presenta due 63x immagini di cellule THP-1, dopo incubazione con anticorpi opsonizzati (verde) e non opsonizzati (rosso) perline, dimostrando la mancanza di assorbimento fagocitaria in assenza di anticorpi. Quando lasso microscopia volta che viene eseguito, l&#39;anticorpo specifico per l&#39;assorbimento fagocitica di perline fluorescenti è ancora più evidente (Movie 1, ingrandimento 20x). Il lavoro precedente ha confermato l&#39;interiorizzazione delle perline associati con le cellule, e gli esperimenti con i monociti primari hanno concordato anche con punteggi fagocitica (valori iMFI) in questo saggio un throughput elevato (dati non riportati). <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="Figura 1"/> Figura 1. Controllo Qualità di analisi. 1A, citometria a flusso istogrammi di fagocitosi per un campione di anticorpi di un soggetto HIV negativo (nero tracce) e un soggetto sieropositivo (traccia grigia).1B: Determinazione sperimentale delle condizioni ottimali di rivestimento tallone per un antigene del campione. Antigene-specifici anticorpi monoclonali (cerchio, quadrato) dimostrano fagocitosi massima di perle rivestite con&gt; 2 mg antigene / l di perline, mentre un anticorpo di controllo (triangolo) dimostra nessuna attività fagocitaria. <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="Figura 2"/> Figura 2. Fagocitosi curva dose-risposta. Campioni clinici degli anticorpi di HIV positivi (trattati, non trattati, e esibendo il controllo della replicazione virale in assenza di terapia antiretrovirale) e HIV negativi fagocitosi guidare soggetti di gp120 (busta HIV) perle di rivestimento differenziato. <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="Figura 3"/> Figura 3. Internalizzazione efficiente. Microscopia conferma l&#39;internalizzazione di anticorpi opsonizzati perline (verde), mentre non opsonizzati perle rimangono in soldistribuzione pesi (ingrandimento 63x). Movie 1. Time-lapse microscopia di anticorpi-driven fagocitosi è stata eseguita nel corso di 14 ore e permette di visualizzare l&#39;attività fagocitaria del THP-1 le cellule utilizzate nel settore high-throughput test. Verde perline fluorescenti sono anticorpi opsonizzati, rosso perline fluorescenti forniscono un controllo negativo. <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">Clicca qui per guardare il film.</a>

<ol>
	<li>Cartron, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+activity+of+humanized+anti-CD20+monoclonal+antibody+and+polymorphism+in+IgG+Fc+receptor+FcgammaRIIIa+gene.">Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcgammaRIIIa gene.</a> Blood. 99, 754-758 (2002).</li><li>Ahmad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+correlation+between+antibody-dependent+cellular+cytotoxicity-mediating+anti-HIV-1+antibodies+and+prognostic+predictors+of+HIV+infection.">Evidence for a correlation between antibody-dependent cellular cytotoxicity-mediating anti-HIV-1 antibodies and prognostic predictors of HIV infection.</a> J. Clin. Immunol. 21, 227-233 (2001).</li><li>Hashimoto, G., Wright, P. F., Karzon, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-dependent+cell-mediated+cytotoxicity+against+influenza+virus-infected+cells.">Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells.</a> J. Infect Dis. 148, 785-794 (1983).</li><li>Anthony, R. M., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th	.">Th	.</a> Curr. Opin. Organ Transplant. , (2010).</li><li>Takai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptors+and+their+role+in+immune+regulation+and+autoimmunity.">Fc receptors and their role in immune regulation and autoimmunity.</a> J. Clin. Immunol. 25, 1-18 (2005).</li><li>Jefferis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+therapeutics:+isotype+and+glycoform+selection.">Antibody therapeutics: isotype and glycoform selection.</a> Expert. Opin. Biol. Ther. 7, 1401-1413 (2007).</li><li>Halstead, S. B., O'Rourke, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dengue+viruses+and+mononuclear+phagocytes.+I.+Infection+enhancement+by+non-neutralizing+antibody.">Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody.</a> J. Exp. Med. 146, 201-217 (1977).</li><li>Subramaniam, K. 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+absence+of+serum+IgM+enhances+the+susceptibility+of+mice+to+pulmonary+challenge+with+Cryptococcus+neoformans.">The absence of serum IgM enhances the susceptibility of mice to pulmonary challenge with Cryptococcus neoformans.</a> J. Immunol. 184, 5755-5767 (2010).</li><li>Ackerman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust,+high-throughput+assay+to+determine+the+phagocytic+activity+of+clinical+antibody+samples.">A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples.</a> J. Immunol. Methods. 366, 8-19 (2011).</li><li>Tsuchiya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1.">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1.</a> Int. J. Cancer. 26, 171-176 (1980).</li><li>Ward, E. S., Ghetie, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effector+functions+of+immunoglobulins:+implications+for+therapy.">The effector functions of immunoglobulins: implications for therapy.</a> Ther. Immunol. 2, 77-94 (1995).</li><li>Winiarska, M., Glodkowska-Mrowka, E., Bil, J., Golab, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+the+antitumor+effects+of+anti-CD20+antibodies.">Molecular mechanisms of the antitumor effects of anti-CD20 antibodies.</a> Front. Biosci. 16, 277-306 (2011).</li><li>Takai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fc+receptors:+their+diverse+functions+in+immunity+and+immune+disorders.">Fc receptors: their diverse functions in immunity and immune disorders.</a> Springer Semin. Immunopathol. 28, 303-304 (2006).</li><li>Yamaguchi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoform-dependent+conformational+alteration+of+the+Fc+region+of+human+immunoglobulin+G1+as+revealed+by+NMR+spectroscopy.">Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy.</a> Biochim. Biophys. Acta. 1760, 693-700 (2006).</li><li>Anthony, R. M., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+the+IgG+Fc+glycan:+the+anti-inflammatory+activity+of+sialylated+IgG.">A novel role for the IgG Fc glycan: the anti-inflammatory activity of sialylated IgG.</a> Fcs. J. Clin. Immunol. 30, S9-S14 (2010).</li><li>Nimmerjahn, F., Anthony, R. M., Ravetch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agalactosylated+IgG+antibodies+depend+on+cellular+Fc+receptors+for+in+vivo+activity.">Agalactosylated IgG antibodies depend on cellular Fc receptors for in vivo activity.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8433-8437 (2007).</li><li>Marchette, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+immune+status+on+dengue+2+virus+replication+in+cultured+leukocytes+from+infants+and+children.">Effect of immune status on dengue 2 virus replication in cultured leukocytes from infants and children.</a> Infect. Immun. 24, 47-50 (1979).</li><li>Fust, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutralizing+and+enhancing+antibodies+measured+in+complement-restored+serum+samples+from+HIV-1-infected+individuals+correlate+with+immunosuppression+and+disease.">Neutralizing and enhancing antibodies measured in complement-restored serum samples from HIV-1-infected individuals correlate with immunosuppression and disease.</a> AIDS. 8, 603-609 (1994).</li></ol>

Nessun conflitto di interessi dichiarati.

Il test qui descritto consente di high-throughput analisi dell&#39;attività fagocitaria dei campioni di anticorpi clinici utilizzando una linea cellulare monocitica lungo utilizzato per studiare i processi di fagocitosi 10 e piastra di citometria a flusso basato automatizzato. Questo test è vantaggioso rispetto ad altri nella sua capacità di caratterizzare con precisione antigene-anticorpi specifici sottoinsiemi, consentendo non solo per lo studio delle differenze di fagocitosi guidato da anticorpi specifici per la malattia da soggetti diversi, ma anche lo studio delle specificità antigeni multipli all&#39;interno della stessa materia. Perché il reclutamento di anticorpi innata cellule effettrici, tra cui monociti e le cellule che presentano l&#39;antigene altri impatti fortemente esito della malattia 11-13, ed è biologicamente geometria variabile a seconda degli anticorpi, sottoclassi di IgG, e glicosilazione a Asparagine297 del dominio Fc 14-16, questo metodo prevede valutazione di un meccanismo di azione degli anticorpi critica. Inoltre, poiché anticorpo-medifagocitosi ated è sfruttata da alcuni agenti patogeni nel corso di infezione da 7,17,18, questo saggio promette nella valutazione del processo di valorizzazione anticorpo-dipendente, e mette in evidenza la duplice natura della fagocitosi come potenzialmente protettivi, così come potenzialmente dannoso l&#39;attività degli anticorpi. Il test descritto può essere utilizzato per analizzare gli anticorpi da campioni clinici di popolazioni di pazienti, ma anche dei vaccinati, e può essere adattato ad utilizzare siero piuttosto che IgG purificata. Inoltre, la secrezione di citochine in risposta alla fagocitosi può essere analizzato dal supernatante della coltura, e l&#39;influenza di FCR specifico può essere determinato utilizzando FCR-bloccanti gli anticorpi, che consente non solo le differenze in potenza fagocitaria da definire, ma a valle di segnalazione eventi, con acume nel meccanismo, e possono aiutare a risolvere e perfezionare la comprensione di questo meccanismo immunitario.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> EZ-Link Micro Sulfo-NHS-LC-biotinilazione Kit </td><td> Thermo Scientfic </td><td> 21935 </td><td></td></tr><tr><td> Amicon Ultra-15 Unità di centrifuga Filtro con membrana Ultracel-50 </td><td> Millipore </td><td> UFC905024 </td><td> Dimensione unità filtro può variare a seconda delle dimensioni antigene </td></tr><tr><td> FluoSpheres NeutrAvidin microsfere marcate, 1,0 micron, giallo-verde fluorescente (505/515) </td><td> Invitrogen </td><td> F-8776 </td><td> I produttori producono questo prodotto in vari colori </td></tr><tr><td> Gel melone Purification Kit IgG </td><td> Thermo </td><td> 45212 </td><td> Si deve prestare attenzione per verificare la purezza dei campioni Ab mediante SDS-page. </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

Anticorpo-driven fagocitosi è indotta attraverso il coinvolgimento dei recettori Fc sulle fagociti professionali, e può contribuire sia spazio così come patologia della malattia. Mentre la proprietà dei domini variabile di anticorpi sono state a lungo considerate critiche per funzione in vivo, la capacità degli anticorpi di reclutare innata cellule immunitarie attraverso i loro domini Fc è diventato sempre più apprezzato come un fattore importante nella loro efficacia, sia nel contesto di ricombinanti La terapia di anticorpi monoclonali, come pure nel corso di infezione naturale o la vaccinazione 1-3. È importante sottolineare che, nonostante la sua nomenclatura, un dominio costante, il dominio anticorpale Fc non ha funzione costante, ed è fortemente modulata dalla sottoclasse di IgG (IgG1-4) e glicosilazione di asparagina 297 4-6. Quindi, questo metodo per studiare le differenze funzionali di antigene-anticorpi specifici in campioni clinici faciliterà la correlazione del piatto fagocitariaziale di anticorpi stato di malattia, suscettibilità alle infezioni, la progressione, o risultato clinico. Inoltre, questa funzione effettrice è particolarmente importante alla luce della documentata capacità degli anticorpi di migliorare l&#39;infezione tramite l&#39;accesso agenti patogeni nelle cellule ospiti attraverso recettore Fc-driven fagocitosi 7. Inoltre, vi sono alcune evidenze che l&#39;assorbimento fagocitaria dei complessi immuni possono influenzare la polarizzazione Th1/Th2 della risposta immunitaria 8. Qui, descriviamo un test progettati per rilevare le differenze di anticorpi indotta fagocitosi, che può essere causato da differenziale sottoclasse IgG, struttura glycan a Asn297, così come la capacità di formare complessi immuni di antigene-specifici anticorpi in un high-throughput di moda . A tal fine, 1 micron perline fluorescenti sono rivestiti con l&#39;antigene, poi incubati con campioni di anticorpi clinici, generando fluorescenti complessi antigene immunitaria specifica. Queste Antibody-opsonizzati perline vengono poi incubate con una linea cellulare monocitica esprimere FcγRs multipli, inclusi sia inibitorio e l&#39;attivazione. Uscita test possono includere attività fagocitaria, la secrezione di citochine, e modelli di utilizzo FcγRs, e sono determinati in modo standardizzato, rendendo questo un sistema molto utile per le differenze di analisi in questo anticorpo-dipendente funzione effettrice in entrambe le infezioni e vaccini-mediata protezione 9.

Watch this Scientific Journal Video about Determining the Phagocytic Activity of Clinical Antibody Samples at JoVE.com

Determining the Phagocytic Activity of Clinical Antibody Samples

Vi presentiamo un elevato throughput saggio citometria a flusso per determinare l&#39;attività fagocitaria dei antigene-anticorpi specifici da campioni clinici, utilizzando l&#39;antigene fluorescenti rivestite perline e una linea cellulare monocitica esprimono più recettori Fc, fornendo utilizzo del recettore e determinazioni attività fagocitaria in una forma standardizzata e moda riproducibile per qualsiasi antigene di interesse.

Determinazione dell&#39;attività fagocitaria dei Campioni anticorpi clinici

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

determining-the-phagocytic-activity-of-clinical-antibody-samples

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Immunology and Infection

23.3K Views.  Ragon Institute of MGH, MIT, and Harvard. Vi presentiamo un elevato throughput saggio citometria a flusso per determinare l'attività fagocitaria dei antigene-anticorpi specifici da campioni clinici, utilizzando l'antigene fluorescenti rivestite perline e una linea cellulare monocitica esprimono più recettori Fc, fornendo utilizzo del recettore e determinazioni attività fagocitaria in una forma standardizzata e moda riproducibile per qualsiasi antigene di interesse.

Video: Determining the Phagocytic Activity of Clinical Antibody Samples

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Determinazione della reattività e Titolo di siero usando un test dell&#39;emoagglutinazione

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

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Immunologia e infezioni

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Utilizzando un pan-virale Assay Microarray (Virochip) allo schermo campioni clinici di patogeni virali

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Rilevazione multiplex di batteri in campioni clinici complessi ed Ambientali con oligonucleotidi accoppiati microsfere fluorescenti

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Determinazione ottimale attività citotossica di specifiche cellule T umane Her2neu CD8 confrontando il Cr51 saggio di rilascio al Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Microscopia a fluorescenza metodi per determinare la vitalità di batteri in associazione con cellule di mammifero

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Scalabile High Throughput Selection Da sintetici Biblioteche Antibody fagi-visualizzato

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una strategia rapido per l&#39;isolamento di nuovi Faustoviruses da campioni ambientali Utilizzando<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

La valutazione rapida e specifica del alogenazione perossidasi attività in campioni di sangue leucociti arricchita

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

La valutazione della retina microglia fagocitaria Funzione<em&gt; In Vivo</em&gt; Usando un test citometria a flusso basato su

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...