We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

ETICA DICHIARAZIONE: L&#39;utilizzo di avanzi di campioni clinici è approvato e segue le linee guida del Memorial Sloan Kettering Cancer Center Institutional Review Board. 1. Prima di eseguire il test NOTA: L&#39;influenza A e B test è approvato per campioni di tamponi nasofaringei e tamponi nasofaringei memorizzati nei mezzi di trasporto virale. I tamponi sono incluse nel kit e devono essere utilizzati per ottenere prestazioni ottimali. Tuttavia, rayon, schiuma, accorrevano tamponi o tamponi nasali poliestere può essere utilizzato anche per raccogliere campioni di tamponi nasali. <ol><li> Per raccogliere un campione di tampone nasale, inserire il tampone nella narice che ha il drenaggio più visibile o che è più congestionato. Con la rotazione dolce, spingere il tampone nella narice fino a incontrare resistenza e ruotare più volte contro la parete nasale prima di rimuovere lentamente dalla narice. Conservare i tamponi e trasportarli in fiale contenenti 3 ml di mezzi di trasporto virale (VTM). NOTA: Una grande attenzione al approprraccolta del campione iate deve essere preso come la raccolta dei campioni inadeguati può produrre risultati errati. NOTA: I campioni su tampone devono essere testati il ​​più presto possibile dopo la raccolta. Tuttavia, essi possono essere conservati a temperatura ambiente per un massimo di 2 ore o in frigorifero a 2-8 ° C per un massimo di 24 ore, se il test non è immediatamente disponibile. esemplari appena raccolti sono l&#39;ideale per ottenere risultati ottimali. Un uso improprio del campione, la conservazione e / o il trasporto possono produrre risultati errati. NOTA: Come misura precauzionale, gli utenti che eseguono questo test dovrebbero trattare tutti i campioni come potenzialmente infettivi seguendo le precauzioni universali durante la manipolazione dei campioni. </li><li> Portare tutti i campioni a temperatura ambiente prima del test. NOTA: Tutte le componenti di prova sono per un solo uso e non devono essere utilizzati per eseguire prove multiple. Non mescolare i componenti del kit con diverso numero di lotto. Non utilizzare i reagenti del kit oltre la data di scadenza. </li><li> Portare il ricevitore del campione azzurro di Rootemperatura m prima del test. La base di test arancione può essere provato senza la necessità di riscaldare a temperatura ambiente. </li><li> Lascia tutti i pezzi di prova nella confezione stagnola fino al momento dell&#39;uso. Accendere lo strumento premendo il pulsante di accensione sul lato dello strumento. Inserire l&#39;user-ID e premere &#39;OK&#39;. </li></ol> 2. Esecuzione del test <ol><li> Per iniziare il processo di test, toccare &#39;Esegui test&#39; sullo schermo dello strumento. Inserire l&#39;ID del paziente utilizzando la tastiera su schermo o uno scanner di codici a barre e premere &#39;OK&#39;. Aprire il coperchio e inserire la base di prova arancione nel supporto di base di test Orange. </li><li> Verificare che il criterio corretto viene visualizzato sullo schermo e premere &#39;OK&#39;. Inserire il ricevitore del campione azzurro nel supporto del ricevitore del campione azzurro. NOTA: Non aprire il ricevitore del campione prima di inserire nello strumento in modo da evitare il tampone di eluizione di raggiungere la corretta temperatura di funzionamento e può influire sulle prestazioni del test. </li&gt; <li> Attendere che il ricevitore del campione si riscaldi. Quando richiesto dallo strumento, togliere la pellicola di sigillo e inserire il tampone del paziente da testare nel ricevitore campione. NOTA: Quando si rimuove la pellicola, posizionare due dita sul bordo del ricevitore di esempio per assicurare che rimanga al suo posto. </li><li> Mescolare energicamente il tampone nel liquido per 10 secondi, premendo il capo tampone contro la parete del ricevitore campione come lo si miscela per aiutare a muovere il campione dal tampone. Premere &#39;OK&#39; per procedere. Se i tamponi di test memorizzati nel mezzo di trasporto virale, VORTEX la VTM per 10 sec quindi aggiungere 200 ml al ricevitore campione. </li><li> Premere la cartuccia di trasferimento bianco nel ricevitore del campione azzurro. Continuare a premere sul ricevitore campione fino la spia arancione aumenta. </li><li> Sollevare e quindi collegare la cartuccia di trasferimento alla base di prova. Osservare l&#39;indicatore arancione scendere una volta che il trasferimento della cartuccia è applicato correttamente. Chiudere il coperchio e non aprire fino a quando il &quot;Controllo completo&quot;Viene visualizzato un messaggio sullo schermo. </li></ol> 3. Controllo Qualità <ol><li> Toccare &#39;Run QC Test&#39; sulla schermata principale. Selezionare il test di controllo di qualità per essere eseguito. Confermare il tipo di test in modo che corrisponda il campione di controllo di qualità destinato al testing toccando &#39;OK&#39; e seguendo le istruzioni visualizzate per completare il test. </li></ol> Interpretazione 4. Risultato <ol><li> Dopo l&#39;esecuzione della prova è completa, osservare i risultati del test visualizzato sullo schermo con interpretazioni per la presenza di influenza A, B, sottotipi sconosciuti. Per ottenere risultati validi ripetere il test. </li></ol> 5. Manutenzione e pulizia <ol><li> Pulire lo strumento e la zona circostante panca ogni giorno con il 70% di etanolo o una soluzione di candeggina al 10%. Spruzzare la soluzione detergente su un panno umido, privo di lanugine per pulire. Non versare le soluzioni direttamente sullo strumento. </li></ol>

<ol>
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The authors have nothing to disclose.

I virus influenzali sono importanti cause mondiali di morbilità e mortalità. diagnosi rapida e accurata di influenza è una delle principali chiavi per la gestione di focolai di influenza durante la stagione delle vie respiratorie. Altri test antigene-based sono rapidi e facili da eseguire; Tuttavia, essi hanno bassi sensibilità 13. D&#39;altra parte, i test molecolari tradizionali hanno migliorato la sensibilità, ma richiedono tecnici di laboratorio più esperti di eseguire e sono più costo...

Infezioni da virus dell&#39;influenza determinano una quantità significativa di morbilità e mortalità ogni anno 1, 2, 3. L&#39;influenza non complicata è caratterizzata da sintomi costituzionali e respiratori quali febbre, mialgia, cefalea e tosse non produttiva 4, 5. Gli individui più anziani, bambini, pazienti immunocompromessi e pazienti con comorbilità sottostanti sono a più alto rischio di complicanze gravi come la polmonite, la miocardite, malattia del sistema nervoso centrale, o la morte 6, 7. L&#39;infezione influenzale è unico da altri virus respiratori in quel tempestiva somministrazione di terapia antivirale entro 48 ore dall&#39;inizio dei sintomi può ridurre la gravità della malattia e la lunghezza 8. La rapida identificazione di influenza è stato anchedimostrato di ridurre l&#39;uso di antibiotici inutili 9, 10. Inoltre, i pazienti ospedalizzati con infezioni influenzali devono essere collocati in locali isolati con opportune precauzioni per il controllo delle infezioni. Tuttavia, le malattie respiratorie causate da virus non influenzali possono essere difficili da distinguere da clinicamente l&#39;influenza. Per questo motivo, i test diagnostici rapidi e precisi per l&#39;influenza è molto importante per la gestione clinica del paziente. Diversi test sono disponibili per il rilevamento e l&#39;identificazione di virus influenzali. Test rapidi di rilevamento dell&#39;influenza antigene (RIDTs) sono ampiamente utilizzati nella pratica clinica come test point-of-care perché sono semplici da usare e fornire i risultati entro 15 a 30 minuti 11, 12; tuttavia, la loro sensibilità variano ampiamente (10-80%) a seconda del produttore e la popolazione in fase di test, e il tipo di influenza e subtype 13, 14, 15. Saggi di fluorescenza diretta (DFA) forniscono sensibilità superiori rispetto RIDTs, ma il tempo di elaborazione è maggiore (~ 3 ore) e devono essere completati da tecnologi esperti 16, 17. Coltura virale è stato il gold standard per la diagnosi di influenza e ha migliorato la sensibilità su entrambi RIDTs e DFA 18. Tuttavia, l&#39;influenza coltura virale può richiedere 2-14 d per completare, diminuendo la sua utilità nel favorire la gestione del paziente 19. Infine, nucleico test di amplificazione degli acidi (NAAT) hanno sostituito tecniche di coltura come il nuovo gold standard nella diagnostica di influenza. NAAT sono considerati avere la massima sensibilità per rilevare l&#39;influenza in poche ore. Tuttavia, incidenza sensibile sono i test più costosi e richiedono attrezzature specializzate e tecnici di eseguire 5 <sup&gt;, 20, 21, 22, 23, 24, 25. Il test dell&#39;influenza A e B qui descritto è il primo test di influenza CLIA-rinunciato molecolare rapida che è prontamente disponibile. Questo test funziona impiegando una reazione intaccare endonucleasi Amplification (NEAR) che utilizza l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Questo test differenzia dell&#39;influenza A da B, richiede 2 min per impostare ed elaborare un campione, e richiede un totale di 15 minuti per completare. Qui, vi presentiamo il protocollo per il saggio Influenza A &amp; B. Inoltre, mettiamo a disposizione un insieme di dati di esempio impostare a confronto le prestazioni del virus dell&#39;influenza A e B saggio sulla archiviato tampone nasofaringeo (NPS) campioni conservati in terreno di trasporto virale (VTM) con un kit pannello patogeno respiratorio.

<table border="0" cellpadding="0" cellspacing="0" width="660">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:231px;">Alere i Instrument</td>
			<td style="width:64px;">Alere</td>
			<td style="width:303px;">NAT-000 (Global), NAT-024 (US)</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Influenza A &#38; B 24 Test Kit</td>
			<td>Alere</td>
			<td style="width:303px;">425-000 (Global), 425-024 (US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere i Barcode Scanner</td>
			<td>Alere</td>
			<td>EQ001001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alere Universal Printer</td>
			<td>Alere</td>
			<td style="width:303px;">55115 (Global), alereiprinter&#160;(US)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L precision pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 &#181;L disposable pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Viral transport medium</td>
			<td>Remel</td>
			<td>M4-RT</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid molecular detection and differentiation of influenza viruses a and b

L&#39;influenza è una malattia respiratoria contagiosa causata da virus influenzali A e B negli esseri umani e provoca una notevole quantità di morbilità e mortalità ogni anno. L&#39;influenza A e B saggio è stato il primo test molecolare rapida influenza CLIA-rinunciato a disposizione. Il test di influenza A e B funziona utilizzando l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Qui, le prestazioni della influenza A e B saggio su congelato, archiviati tampone nasofaringeo (NPS) campioni conservati in terreno di trasporto virale (VTM) sono stati confrontati con un test del pannello respiratoria. Le prestazioni della A e B saggio Influenza stata valutata confrontando i risultati al metodo di riferimento del pannello respiratoria. La sensibilità per il virus dell&#39;influenza A totale è stata del 67,5% (95% CI (CI), 56,6-78,5) e la specificità del 86,9% (CI, 71,0-100). Per il test virus dell&#39;influenza B, la sensibilità e la specificità sono state 90,2% (CI, 68,5-100) E il 98,8% (CI, 68,5-100), rispettivamente. Questo sistema ha il vantaggio di un tempo di prova significativamente più breve rispetto a qualsiasi altro test molecolare attualmente disponibili e la procedura semplice, priva di pipetta viene eseguito su un chiuso, sistema completamente integrato dimensioni ridotte. Nel complesso, l&#39;influenza A e dosaggio B valutati in questo studio ha il potenziale per servire come test diagnostico point-of-care rapida influenza.

In questo studio, i campioni NPS archiviati sono stati raccolti da pazienti ricoverati con sintomi simil-influenzali al Memorial Sloan-Kettering Cancer Center (MSKCC) nel corso di un focolaio di influenza tra il 15 Dicembre, 2012 e il 1 marzo 2013. I campioni NPS sono state presentate in 3 ml di VTM e testato come parte della pratica clinica di routine con un saggio molecolare che rileva un gruppo di virus respiratori (RP), tra cui l&#39;influenza a, a-1, a-3, e B. Durante il periodo di ...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Descriviamo un test rapido, molecolare a base di Influenza A e B. Il saggio Influenza rileva ogni bersaglio entro 15 minuti utilizzando l&#39;amplificazione isotermica con primer specifici per l&#39;influenza seguita da rilevamento di target con sonde segnale molecolare. Il saggio e influenza A B è facile da usare e richiede minimi hands-on tempo per eseguire.

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

rapid-molecular-detection-differentiation-influenza-viruses

Research

JoVE Journal

Immunology and Infection

15.5K Views.  Memorial Sloan Kettering Cancer Center.  Descriviamo un test rapido, molecolare a base di Influenza A e B. Il saggio Influenza rileva ogni bersaglio entro 15 minuti utilizzando l'amplificazione isotermica con primer specifici per l'influenza seguita da rilevamento di target con sonde segnale molecolare. Il saggio e influenza A B è facile da usare e richiede minimi hands-on tempo per eseguire. 

Video: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

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

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

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

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

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

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

Ricerca

Immunologia e infezioni

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX rilevazione colorimetrica su un DNA microarray per l&#39;influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

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

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

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

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

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Rilevazione di True IgE-esprimono mouse B Cells Lineage

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Panni nasale per l&#39;influenza A rilevazione antivirus e isolamento da Suina

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Rilevamento e l&#39;arricchimento di Rare antigene-specifiche cellule B per l&#39;analisi del fenotipo e funzione

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Rilevazione rapida molecolare e la differenziazione dei virus influenzali A e B

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Capitoli in questo video

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

ampliPHOX rilevazione colorimetrica su un DNA microarray per l'influenza

High-throughput Metodo di rilevazione per il virus dell'influenza

Analisi rapida di aberrazioni cromosomiche in linfociti B mouse da PNA-FISH

Rilevazione di True IgE-esprimono mouse B Cells Lineage

Intranasale somministrazione di ricombinante Influenza vaccini nei modelli chimerico topo per studiare immunità delle mucose

Panni nasale per l'influenza A rilevazione antivirus e isolamento da Suina

Rilevamento e l'arricchimento di Rare antigene-specifiche cellule B per l'analisi del fenotipo e funzione

Basato sulla fluorescenza neuraminidasi Inibizione Assay per valutare la suscettibilità dei virus influenzali di neuraminidasi Inhibitor classe di antivirali

Neutralizzando le risposte dell'anticorpo al virus A(H3N2) in sieri umani di Microneutralization l'Influenza di misurazione analisi utilizzando cellule MDCK-SIAT1