Biz Virochip protokolün ilk geliştirme ve optimizasyonu için David Wang, Anatoly Urisman Amy Kistler, Kael Fischer, Patrick Tang, ve Alexander Greninger teşekkür ederim. Bu çalışma bir NIH K08 hibe ve Abbott Keşif Ödülü (CC) ve Howard Hughes Tıp Enstitüsü (JLD) tarafından desteklenmektedir.

Virochip testinin adımlar (1) klinik örneklerden nükleik asit ekstraksiyon, (2) çıkarılan RNA ters transkripsiyon ve 2. iplikçik cDNA sentezi, rastgele astarlanmalıdır cDNA (3) PCR, (3) Cy3 floresan boya dahil (4) Virochip mikroarray etiketli malzeme hibridizasyon ve (5) tarama ve analizi (Şekil 1). Aşağıda gösterilen protokol grip benzeri bir hastalığa sahip bir çocuğun bir nazal sürüntü örneği Virochip testinin kullanımını göstermek. Primer Diziler Primer bir 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B 5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Nükleik Asit Ekstraksiyonu <ol><li> Bu örnekleri potansiyel bulaşıcı olarak klinik örneklerden nükleik asit ekstraksiyon, Biyolojik Güvenlik Seviye 2 (BSL-2) veya daha yüksek koşulları altında yapılmalıdır. </li><li> Nazal sürüntü örneği viral tutarak orta taşınır. 1.5 ml Eppendorf mikrosantrifüj tüp içine örnek kısım 200 mcL. </li><li> İsteğe Bağlı: 0.22 mikron filtre (viral parçacıkların arındırmak için) geçmesine örnek </li><li> İsteğe bağlı: Turbo DNAz kiti (Ambion) kullanarak üreticinin talimatlarına göre DNAz (ana bilgisayar genomik DNA aşağılamak ve arındırmak için korumalı, encapsidated viral nükleik asit) ile ön işlemden örnek </li><li> Zymo ZR viral RNA Ekstraksiyon Kiti veya üreticinin talimatlarına göre Qiagen Ultrasens Virus Kit kullanarak örnek ayıklayın. </li></ol> 2. Ters Transkripsiyon ve 2.-iplikçik cDNA sentezi (&quot;Yuvarlak A&quot;) <ol><li> 4 uL çıkarılan RNA 1 uL 40 mcL / pmol Primer ekleyin. Isı 65 ° C PCR (örneğin GeneAmp PCR Sistemi 9700) ve daha sonra 5 dakika boyunca oda sıcaklığında x 5 dakika soğumasını bekleyin. </li><li> 2 mcL 5X RT tampon, 1 mcL 12.5 mM dNTP, 1 mcL su, 0.5 mcL 0.1M DTT, ve 0.5 mcL SSIII RT (Invitrogen) oluşan bir ana karışımı 5 mcL ekleyin. 42 ° C&#39;de x 60 dakika. </li><li> Programlanabilir bir PCR, ısı 94 ° 10 serin C x 2 dk (iptal etmek için ters transkriptaz reaksiyonu), ° C, nabız santrifüj, ve ardından 1 mcL 5X Sequenase tampon, 3.8 mcL su oluşan 5 mcL Sequenase karışımı ekleyin, 0.15 mcL Sequenase (USB Corporation). 2. iplikçik sentez için, 10 ° C - 37 ° C 8 dk boyunca, 94, 8 dakika sonra, ısı tutun ° C x 2 dk (Sequenase inaktive etmek için) ve 10 ° C (basılı tutun) serin kadar rampa </li><li> İsteğe bağlı: Rd örnek bir cDNA -20 ° C&#39;de bu noktada saklanabilir </li></ol> 3. PCR Amplifikasyon rastgele Astarlı cDNA (&quot;Yuvarlak B&quot;) <ol><li> 5 Rd uL bir örnek, 45 mcL 5 mcL 10X PCR buffer, 1 mcL 12.5 mM dNTP, 1 mcL 100 pmol / mcL astar B, 1 mcL KlenTaq LA (Sigma), ve 37 mcL su oluşan bir ana karışımı ekleyin . </li><li> PCR protokolü aşağıdaki gibi çalıştırın: 94 ° C, 2 dakika → (25 devir 94 ° C, 30 / 50 ° C, 45 s / 72 ° C, 1 dak.) → 72 ° C, 5 dk → 10 ° C ( basılı tutun) </li><li> % 1.5 agaroz elektroforez jel üzerinde Rd B numunesi kontrol edin. A smear yaklaşık 200 - 1000 bp görüntülenmeyecektir gerekir (Şekil 2A) </li></ol> 4. Cy3 Floresan Boya Kuruluş <ol><li> İsteğe Bağlı: Cy5, floresan boya ile başka bir örnek etiketli olabilir ve aynı mikroarray için melezleşmiştir. </li><li> Aa-dNTP birleştirmek için, 5 uL 10X PCR buffer, 1 uL 12.5 mM aa-dNTP (Invitrogen), 1 uL 100 pmol / uL astar Yatak, 1 oluşan bir ana karışımı 45 uL Rd B numunesinin 5 uL 1 uL KlenTaq LA (Sigma), ve 37 uL su. PCR protokolü aşağıdaki gibi çalıştırın: 94 ° C, 2 dakika → (15 döngü 94 ° C, 30 / 50 ° C, 45 s / 72 ° C, 1 dak.) → 72 ° C, 5 dk → 10 ° C ( basılı tutun) </li><li> Rd C örnek üreticinin talimatlarına göre DNA Temiz ve Konsantratörü-5 Seti (Zymo Araştırma) ile temizleyin. 10 mcL Zehir. </li><li> 1M bikarbonat 1 uL (Sigma) ve Rd C Örnek 1 uL Cy5 (GE Healthcare) ekleyin. </li><li> Inkübe x 1 saat karanlıkta. </li><li> Üreticinin talimatlarına göre DNA Temiz ve Konsantratörü-5 Seti (Zymo Araştırma) ile Cy3 etiketli örnek temizleyin. 12 mcL Zehir. </li></ol> 5. Hibridizasyon için Virochip Mikroarray <ol><li> İsteğe bağlı: (numune normalleşmesi için), Nanodrop spektrofotometre dahil cDNA konsantrasyon ve boya miktarını kontrol 1.5 mcL </li><li> 1 mcL 25X parçalanma tampon, 5 mcL 5X engelleme tampon, 9.5 mcL Cy3-etiketli örnek (veya normale miktarı), su ve PCR şeridi tüpü 25 mcL toplam hacmi (Agilent) eklemek </li><li> 2X GEX hibridizasyon tamponu (Agilent) 25 mcL ekleyin. Kabarcıkları kısaca aşağı spin. </li><li> Conta slayt üzerine numune 40 mcL yükleyin. </li><li> Place Agilent baskılı Virochip array (Californi Üniversitesi, San Francisco / Agilent) (aşağı &quot;Agilent&quot; işaretli tarafı) ve conta slayt üst sıkıca vidayı sıkın. </li><li> 65 gecede melezleşme ° C fırında 10 devirde hız ayarı. </li><li> 37 ° C&#39;ye kadar diziler, ön ısıtma ve ön tampon 2 (Agilent) yıkamak için Tampon 1 (Agilent) conta slayt / dizi sandviç sualtı ayrı ele alalım. Tampon 1 x 1 dakika yıkayın. Önceden ısıtılmış tampon 2 x 1 dakika yıkayın. Yavaş yavaş, kabarcıkları önlemek için dikkatli olmak (kimwipe uzakta doğrudan dizi aktif yüzlerine dokunmaktan kaçınmak için dikkatli olmak ekstra nem fitil kullanın), tampon 2 slayt kaldırmak </li><li> Slayt slayt tutucunun içine yerleştirin ve dizi tarayıcı takın. </li></ol> 6. Virochip Tarama ve Analizi <ol><li> Tarama Cy3 etiketli dizi araç standart tarama protokolü (Şekil 2B) göre 5 mm veya 2 mm. </li><li> Virochip mikroarray&#39;ler görsel denetim, kümeleme analizi, ya da otomatik Virochip analiz programı tarafından analiz, 11 E-tahmin edin . Bu üç yöntemin her biri tarafından, grip virüsü burun sürüntü örneği (influenza benzeri hastalığı olan bir çocuk) (Şekil 2C) kolayca tespit </li></ol> 7. Temsilcisi Sonuçlar: Protokol doğru yapıldığında, bir karalama Rd B adım (Şekil 2A) sonra agaroz jel elektroforez görülebilir. Agilent platformu Virochip mikroarray (Şekil 2B) görsel olarak analiz etmek zor olacak, ama bir virüs varsa kolayca görsel denetim, kümeleme analizi, ve / veya E-Tahmin (Şekil 1C) tespit edilmelidir. Tayini için bir pozitif kontrol olarak hizmet; Numuneler bilinen bir virüs (MS2 bakteriyofaj örneğin tütün mozaik virüsü) nükleik asit ölçülen miktarları ile çivili olabilir. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Şekil 1" /> Şekil 1. Virochip mikroarray işleme ve analiz şematik klinik örneklerden nükleik asit Çıkarılan rastgele amplifiye floresan boya ile işaretlenmiş ve Virochip mikroarray için melezleşmiştir. Mikroarray&#39;ler 2 mikron çözünürlükte taranır ve E-tahmin de dahil olmak üzere, çeşitli hesaplama araçları kullanılarak analiz edildi. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Şekil 2" /> Şekil 2. Virochip tayininde Adımlar amplifikasyon sonra rastgele PCR, bir karalama 200 - 1000 bp jel elektroforezi ile görüntülendi (A) (B) Üç Virochip mikroarray&#39;ler 8 diziler / cam slayt küçük bir bölge ile gösterilir. (C) sağ alt köşesinde, içeride havaya uçurulan bir mikroarray Otomatik mikroarray viral analizi ile klinik örnek, influenza A virüsünün varlığını gösteren E-tahmin edin. Yüksek benzerlik skoru (s = 0.55), bu nedenle bu son derece önemli bir tahmin yapmak, sıfıra yakın bir p-değeri karşılık gelir.

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	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

Virochip protokolü burada açıklandığı gibi karmaşık ve yetenekli ve titiz bir araştırma teknisyeni gerektirir. Doğru reaktif konsantrasyonu ve PCR etiketleme koşulları ve hibridizasyon kritik öneme sahiptir. Virochip protokolü nükleik asit ekstraksiyon TRIzol (Invitrogen) gibi bir doku çıkarma yönteminin kullanımı ile hastalıklı dokuların analizi kolayca uyum sağlamak için değiştirilebilir. Problar eklendi veya istenen spektrumu ve kapsamının genişliği elde etmek için gerekli silinmiş olabilir, örneğin &quot;-the-fly&quot;, bakteri ve mantarlar gibi nonviral hedeflerin saptanması için yoklamaları dizayn edilebilir ve ekledi. Henüz geliştirme aşamasında Virochip testinin bazı uygulamalar, klinik laboratuvar, salgın araştırma, saflık taraması, ilaç ve aşılar ve yeni viral patojen keşif geniş spektrumlu bir viral teşhis içerir.

<table border="1"><tbody><tr><th> Reaktif / malzeme Adı </th><th> Reaktif / malzeme Adı </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA (N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Master Mix (5 mcL) </td><td> 2 mcL 5X RT tampon </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL su </td></tr><tr><td> 0.5 mcL 0.1M DTT </td></tr><tr><td> 0.5 mcL SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 mcL) </td><td> 1 mcL 5X Sequenase tampon </td></tr><tr><td> 3.8 mcL su </td></tr><tr><td> 0.15 mcL Sequenase (USB Corporation&#39;ın) </td></tr><tr><td rowspan="5"> Rd B PCR Master Mix (45 mcL) </td><td> 5 mcL 10X PCR buffer </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL 100 mcL / B pmol astar </td></tr><tr><td> 1 mcL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mcL su </td></tr><tr><td rowspan="5"> Rd C PCR Master Mix (45 mcL) </td><td> 5 uL 10X PCR buffer </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL 100 mcL / B pmol astar </td></tr><tr><td> 1 mcL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mcL su </td></tr><tr><td rowspan="4"> Hibridizasyon tamponu (25 mcL) </td><td> 1 mcL 25X parçalanma tampon (Agilent) </td></tr><tr><td> 5 mcL 5X engelleme tamponu (Agilent) </td></tr><tr><td> Cy3-etiketli örnek (veya normale miktarı 9.5 mcL) </td></tr><tr><td> 25 mcL toplam hacim su ekleyin. </td></tr><tr><td> Agilent Virochip mikroarray </td><td> Chiu laboratuvar, UCSF edinilebilir; bekleyen lisans </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

Virüslerin doğal dizisi çeşitliliği yanı sıra, geleneksel yöntemlerle algılanamaz SARS coronavirüs ve 2009 pandemik H1N1 influenza virüsü gibi roman viral patojenler, devam eden ortaya çıkması nedeniyle, pek çok bulaşıcı hastalıkların viral nedenlerin tanısı zordur. Bu zorlukları ele almak için, daha önce geliştirilmiş ve kapasitesi ile korunmuş sıra homoloji 1 dayanarak, bilinen tüm virüsleri yanı sıra roman varyantları tespit etmek için Virochip denilen bir pan-viral mikroarray platformu valide . Virochip kullanarak, tam spektrum, eşdeğer ya da geleneksel klinik test 2-5 üstün bir hassasiyet ile hastanede yatan hastalarda açıklanamayan kritik hastalık vakaları da dahil olmak üzere, solunum yolu enfeksiyonları ile ilişkili virüs tespit ettik. Virochip ayrıca, SARS coronavirüs 6,7, bir roman rinovirüs dalının 5, XMRV (prostat kanseri ile bağlantılı bir retrovirüs) 8, kuş bornavirus (papağanlar bir israf hastalığı nedeni) 9, dahil olmak üzere, yeni virüsleri tanımlamak için kullanılır olmuştur solunum ve ishal hastalık 10 ile çocuklarda ve yeni bir cardiovirus. Virochip güncel sürümü Agilent mikroarray platforma taşıdık ve ~ 36.000 probları Aralık 2009 itibariyle GenBank ~ üzerinde 1.500 virüslerden kaynaklanan oluşur. Burada örnek nükleik asit ekstraksiyon, rastgele primerler kullanılarak PCR amplifikasyonu, floresan boya dahil, ve mikroarray hibridizasyon, tarama ve analiz de dahil olmak üzere, (~ 24 saat gerçekleştirme süresi) bitirmek için baştan bir Virochip testi işleme dahil adımları ortaya koymaktadır.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

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

Virochip korunmuş dizi homoloji temelinde aynı anda, bilinen tüm virüsleri yanı sıra roman virüsler tespit etmek için tasarlanmış bir pan-viral mikroarray. İşte biz, varlığı bilinen ve bilinmeyen virüslere hem de klinik örnekleri analiz etmek için bir Virochip tahlil nasıl çalıştırılacağını göstermektedir.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

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 ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

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

18.8K Görüntüleme.  University of California, San Francisco. Virochip korunmuş dizi homoloji temelinde aynı anda, bilinen tüm virüsleri yanı sıra roman virüsler tespit etmek için tasarlanmış bir pan-viral mikroarray. İşte biz, varlığı bilinen ve bilinmeyen virüslere hem de klinik örnekleri analiz etmek için bir Virochip tahlil nasıl çalıştırılacağını göstermektedir.

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

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Klinik Antikor Örnekleri fagositik Aktivitesi Belirlenmesi

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

İmmünoloji ve Enfeksiyon

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Bir Tam Kan fenotipleme Testi Kullanarak Çok Merkezli Klinik Araştırmalar için Binbaşı Periferik Kan Lökosit Populasyonlarının Numaralama

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Ana-Hücre RNA sentezi üzerinde Viral Enfeksiyon Etkisi Tedbir tıklayın Kimya Kullanımı

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Floresan Proteinleri kullanarak Afrika tripanosom içinde Glycosome Dynamics Monitör

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A Virüsü hemaglutininler aviditesi ve özgünlüğünün değerlendirilmesi için Minyatür Glycan Mikroarray Testi

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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.

Kullanımı Çevre Örneklerinde Yeni Faustoviruses İzolasyonunda bir Hızlı Stratejisi<em&gt; Vermamoeba vermiformis</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 ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Enzim-linked Lektin Testi tarafından Grip Nöraminidaz İnhibisyon antikor titreleri Ölçme

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

Viral Örneklerin Negatif Boyanması İçin Kapsüllerin Biyokontainment İçerisinde Kullanımı

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

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