Yazarlar NIH 3R01AI080289-02S1 ve Harvard Üniversitesi Center NIH / NIAID 2P30AI060354-07 altında AIDS Araştırma Görevlisi Bursu için bir fon kabul etmek istiyoruz.

1. Kültür fagositik hücreler <ol><li> Kültür THP-1 10 hücre RPMI 1640 T matara sabit bir süspansiyon olarak% 10 fetal sığır serumu ile desteklenmiştir. FcγR ifade ve test performansı tutarlı düzeyde korumak için hücre yoğunluğu 0.5x10 6 / ml altında tutulmalıdır. </li></ol> 2. Biotinlenmiş Antijen hazırlayın <ol><li> Üreticinin talimatlarına göre faiz hedef antijen biotinylate için gerekli sulfo-NHS LC biotin reaktif kiti miktarını hesaplayın. </li><li> Biyotinilasyon reaktif su içinde çözülür ve birincil aminler içermez bir tampon hemen antijen için hesaplanan miktar ekleyin. Reaksiyon, ara sıra karıştırarak, oda sıcaklığında 1 saat süreyle devam etmek için izin verin. </li><li> Küçük kalkan korumak için uygun bir molekül ağırlığı kesme Amicon santrifüj konsantrasyon biriminde tampon döviz fazlalığı, indirekt biotin çıkarınt antijen. Konsantrasyon birimi üst odasına örnek ekleyin ve toplam hacmi 15 ml doldurma çizgisine getirmek için PBS ekleyin. Yaklaşık 1.5 ml hacim getirmek için 4000 xg&#39;de santrifüjleyin. Bu işlemi tekrarlayarak boncuk antijen maksimum kaplama sağlamak için iki kez serbest biotin% 99 kaldıracaktır. </li></ol> 3. Antijen Doymuş Boncuk hazırlayın <ol><li> Iki kere 1, 1 mm floresan neutravidin boncuk 100 ul yıkayın ml% 0.1 PBS-BSA yüksek hızda bir mikrosantrifüj iplik sonra. Yeniden süspanse 10 tüp içine 100 ul PBS-BSA ve kısım boncuk yıkanır. </li><li> Boncuklar doyurabilecek antijen kaplama koşulları belirlemek için, iki kat adımlar biotinlenmiş antijen konsantrasyonlarında değişen yıkanmış boncuk süspansiyonu ile 10 ul birleştirir. Rotator mikrosantrifüj tüp 4 ° C&#39;de bir gece inkübe edin. </li></ol> NOT: Doygunluk boncuk, deneysel olarak tespit edilmelidir. Bu yapılabilir, boncuk, sonradan kontrol monoklonal antikor ile opsonized zaman maksimum fagositozda verimi boncuk kaplama koşulları belirleyerek. <ol start="3"><li> Boncuk pelet kadar yüksek hızda 1 ml PBS-BSA ve santrifüj ile yıkanarak bağlanmamış antijen çıkarın. PBS-BSA kaldırın ve tekrar edin. </li><li> Son hacim 1 ml PBS-BSA yeniden süspanse antijen kaplı boncuk. Boncuk 4 ° C kullanımdan önce bir hafta kadar saklanır olabilir. </li></ol> 4. Antikor Örnekleri hazırlayın <ol><li> Klinik antikor örnekleri, plazma, üretici talimatlarına göre bir Kavun jel IgG saflaştırma kiti kullanarak arındırılmalıdır. Saflaştırılmış IgG 4 ° C kullanıma hazır olana kadar saklanabilir. </li></ol> NOT: Uygun önlemler işlenmesiyle ilgili insan numunelerinde her zaman alınmış gerekir. <ol start="2"><li> A280 absorbans antikorlar arıtılmış konsantrasyonu belirlemek ve örnekleri PBS içinde 1 mg / ml seyreltik. </li><li> Varsaymak hazırlayın1 mg / ml PBS seyreltme ive ve negatif monoklonal kontrol antikorlar. </li></ol> 5. Deney Kaplama <ol><li> Vorteks yukarıda hazırlanan yıkanmış, antijen doymuş boncuk çözüm süspanse edin ve yuvarlak bir alt 96 plaka her kuyuya 10 ul aktarmak. Bakım, her bir kuyunun eklenir eşit sayıda floresan boncuk sağlamak için sürekli boncuk süspansiyonu ajitasyon alınmalıdır. </li><li> Her antikor için bir doz-yanıt eğrisi oluşturma, her bir kuyunun ilgi antikorlar konsantrasyonları değişen ekleyin. Optimal konsantrasyonları mevcut antikor titresi bağlı örnekleri arasında farklılık gösterir, ancak 0.01-100 mikrogram / ml final konsantrasyonda bir dizi iyi kapsama sağlamak ve ilgi konsantrasyon aralığı belirlenmesini sağlayacaktır. 20 ul daha geniş hacimli olun antikorları eklenir. </li><li> 37 2 saat inkübe boncuk ve antikor örnekleri ° C antikorları boncuk opsonize için izin vermek. &lt;/ Li&gt; <li> THP-1 hücrelerinin 2.5 x 10 5 hücre / ml süspansiyon hazırlamak, toplam 5 x, her bir kuyunun bu süspansiyon 200 ul 10 4 THP-1 hücreler her iyi. </li><li> 37 ° gecede ° C&#39;de,% 5 sabit bir inkübatör CO 2, yerçekimi üzerinden pelet izin hücreleri ve boncuk. </li></ol> NOT: Sinyal gürültü optimal boncuk belirlenmesinde tarafından geliştirilmiş olabilir: antikoru: THP-1 hücre oranları belirli bir antijen ve antikor kaynağı için. 6. Akış sitometrik analizi <ol><li> Her hücre pelletini rahatsız etmemek için dikkatli olmak süpernatant 100 ul çıkarın. Sitokin salgılanması tespitler veya diğer analizler istendiğinde supernatant kaydedilebilir. </li><li> Sabitlenmesi için, her bir kuyu ve pipetlemeyin hücreleri tekrar süspansiyon ve mix 100 ul% 4 paraformaldehid ekleyin. </li><li> Yüksek verim akım sitometri analizi bir HTS plaka okuyucu ile donatılmış bir BD LSR II kullanılarak yapılabilir.</li><li> Program yazılımı her iyi üç kez (100 ul karışımın hacmi) karıştırın ve her numunenin 30 ul, ya da en az 2000 hücre olaylar, analiz etmek. </li><li> FlowJo veya eşdeğer yazılım toplanan veriler analiz edilebilir. Faydalı ölçümlerini boncuk + yüzde floresan hücreler, mevcut fagositik hücrelerin sayısının bir ölçüsüdür sağlar, fagosite boncuk sayısının bir ölçüsüdür sağlar fagositik hücrelerinin yanı sıra floresan içerir. Bu değerlerin çarpılması entegre bir ortalama floresan yoğunluğu, veya iMFI üretir. </li><li> Her fagositik hücre tarafından fagosite boncuk ortalama sayısı 1 boncuk ortalama floresan iMFI bölünmesi ile hesaplanır. </li></ol> 7. Temsilcisi Sonuçlar Etkilenen ve etkilenmeyen konularda antikor örneklerinin açık bir farklılaşma mevcut olmalıdır. Şekil 1A, HIV negatif bir antikor örnek FACS histogramlartive (siyah eser) ve HIV pozitif (gri iz) konu ve antijen-spesifik antikorların varlığı ile tahrik artan fagositoz göstermektedir. Optimum duyarlılık testinin biotinlenmiş antijen ile boncuk doygunluğu bağlıdır. Şekil 1B farklı miktarlarda antijen ile kaplı boncuklar (bağlayıcı olmayan bir antikor, üçgenler de dahil olmak üzere) 3 kontrol monoklonal antikorlar ile opsonized olduğu gözlenen fagositoz sunar ve bir doyurarak konsantrasyon olarak bu antijeni 2μg antijen / ml boncuk kurar. Bir doz-yanıt eğrisi Şekil 2&#39;de sunulan ve 0,05-5 mg / ml antikor konsantrasyon aralığında fagositoz ikna etmek için tabi antikor örneklerinin diferansiyel kapasitesini gösteriyor. Bu diferansiyel fagositoz titresi veya IgG alt ve glikozilasyon devlet olarak Fc etki alanı özellikleri farklılıklar ya da tahrik olabilir. THP-1 hücreleri i zamanMaged floresan mikroskobu ile, boncuk fagositoz açık bir kanıt yoktur. Şekil 3, antikor opsonized (yeşil) ve non-opsonized (kırmızı) boncuk ile inkübasyon sonra antikor yokluğunda fagositik alımı eksikliği gösteren THP-1 hücreleri hala görüntüleri iki 63x sunar. Zaman atlamalı mikroskopi yapıldığında, floresan boncuk antikor özel fagositik alımı daha da çarpıcıdır (Film 1, 20x büyütme). Önceki iş hücreleri ile ilişkili boncuk içselleştirilmesi onaylamıştır ve birincil monositler ile ilgili deneyler, bu yüksek verim assay (veriler gösterilmemiştir) fagositik puanları (iMFI değerler) ile mutabık kalmışlardır. <img src="/files/ftp_upload/3588/3588fig1.jpg" alt="Şekil 1"/> Şekil 1 Kalite Kontrol Testi. 1A, bir antikor örnek için HIV negatif bir konu (siyah iz) ve HIV pozitif bir konuda (gri iz) fagositoz Flow sitometri histogramlar.1B: Deneysel örnek bir antijen için en uygun boncuk kaplama koşullarının belirlenmesi. Bir kontrol antikor (üçgen) fagositik aktivite gösterir antijen-spesifik monoklonal antikorlar (daire, kare), boncuk antijen / ul&gt; 2 mg ile kaplı boncuk maksimum fagositoz göstermektedir. <img src="/files/ftp_upload/3588/3588fig2.jpg" alt="Şekil 2"/> Şekil 2 Fagositoz doz-yanıt eğrisi. Farklı HIV pozitif (tedavi edilen, işlenmemiş, viral replikasyon kontrolü ve anti-retroviral tedavi yokluğunda sergileyen) ve gp120 HIV negatif konular sürücü fagositoz (HIV zarf) kaplı boncuk Klinik antikor örnekleri. <img src="/files/ftp_upload/3588/3588fig3.jpg" alt="Şekil 3"/> Şekil 3. Verimli İçselleştirme. Mikroskopi, non-opsonized boncuk sol kalırken antikor opsonized boncuk (yeşil) içselleştirilmesi teyitution (63x büyütme). Movie 1, antikor-odaklı fagositoz Time-lapse mikroskopi 14 saat boyunca yüksek verim tayininde kullanılan THP-1 hücrelerinin fagositik aktivite görselleştirme yapılır ve izin verir oldu . Yeşil floresan boncuk antikor opsonized, kırmızı floresan boncuk olumsuz bir kontrol sağlar. <a href="http://www.jove.com/files/ftp_upload/3588/3588movie1.mov">film izlemek için buraya tıklayın.</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>

Çıkar çatışması ilan etti.

Burada açıklanan tahlil fagositik süreçleri 10 ve plaka bazlı otomatik flow sitometri uzun çalışma için kullanılan bir monositik hücre hattı kullanılarak klinik antikor örneklerinin fagositik aktivitesi yüksek verimlilik analizi sağlar. Bu testte sadece farklı konularda hastalığa özgü antikorlar tarafından tahrik fagositoz farklılıklar çalışma için izin, antijen-antikor altkümelerini tam olarak karakterize etmek için kendi yeteneği diğerleri üzerinde avantajlı değil, aynı zamanda, aynı konuda içinde birden fazla antijen özgüllükleri çalışma. Antikor işe doğuştan efektör hücreleri, monositler ve diğer antijen sunan hücreler de dahil olmak üzere güçlü bir hastalık sonucu 11-13 etkileri ve Fc etki alanı 14-16 Asparagine297 antikor geometri, IgG alt ve glikozilasyon bağlı olarak biyolojik değişken olduğu için, bu yöntem sağlar. kritik bir eylem antikor mekanizmasının değerlendirilmesi. Ayrıca, antikor-medi çünküated fagositoz 7,17,18 enfeksiyonu seyri sırasında bazı patojenler tarafından sömürüldü, bu testte antikor-bağımlı geliştirme sürecinin değerlendirilmesi söz tutan ve potansiyel koruyucu, hem de potansiyel olarak zararlı olarak fagositoz ikili doğasını vurgular antikor aktivitesi. Açıklanan test hasta popülasyonları klinik örneklerinden antikorlar analiz etmek için kullanılan, aynı zamanda aşılananların ve saflaştırılmış IgG yerine serum kullanmak için adapte edilebilir olabilir. Ayrıca, kültür süpernatant fagositoz yanıt sitokin salgılanması analiz edilecek ve özel FCR etkisi belirlenecek fagositik potens tek fark sağlayan, FCR-bloke edici antikorlar kullanarak tespit edilebilir, ancak aşağı akım anlayışı ile, olaylar sinyalizasyon mekanizmasını çözmek ve bu bağışıklık mekanizmasının anlaşılmasını ve rafine yardımcı olabilir.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Micro Sulfo-NHS-LC-biyotinilasyon Kit EZ-Link </td><td> Thermo Scientfic </td><td> 21935 </td><td></td></tr><tr><td> Ultracel-50 membran ile Amicon Ultra-15 Santrifüj Filtre Ünitesi </td><td> Millipore </td><td> UFC905024 </td><td> Filtre birim boyutu antijen boyutuna göre değişebilir </td></tr><tr><td> FluoSpheres NeutrAvidin mikroküreler, 1.0 mm, sarı-yeşil floresan (505/515) etiketli </td><td> Invitrogen </td><td> F-8776 </td><td> Üreticiler, çeşitli renklerde bu ürün üretmek </td></tr><tr><td> Kavun Jel IgG Arıtma Kiti </td><td> Thermo </td><td> 45212 </td><td> Ab örneklerin SDS-sayfası saflığını doğrulamak için alınmalıdır. </td></tr></tbody></table>

determining the phagocytic activity of clinical antibody samples

Antikor-odaklı fagositoz nişan profesyonel fagositler Fc reseptörleri aracılığıyla ortaya çıkar ve hastalığın yanı sıra her iki boşluk patoloji katkıda bulunabilir. Antikorların değişken etki özellikleri uzun in vivo fonksiyonu için kritik olarak kabul edilmiştir, antikor Fc etki alanları üzerinden doğuştan gelen bağışıklık hücreleri işe yeteneği hem rekombinant bir ortamda, önemli bir faktör olarak etkinliğini giderek takdir haline gelmiştir monoklonal antikor tedavisi yanı sıra, doğal enfeksiyon sırasında ya da 1-3 aşı . Önemlisi, sürekli bir etki alanı olarak isimlendirilmesi rağmen, antikor Fc, etki alanı sabit fonksiyon ve IgG alt sınıf (IgG1-4) ve glikozilasyon 297 4-6 Asparagin az güçlü modüle değildir . Böylece, bu yöntem klinik örneklerde antijen-spesifik antikorların işlevsel farklılıklar çalışma fagositik pot korelasyon kolaylaştırmakantikorların hastalık durumu, enfeksiyona yatkınlık, ilerlemesi, ya da klinik sonuç ential. Ayrıca, bu efektör fonksiyon Fc reseptör odaklı fagositoz 7 ile konak hücre içine patojenler erişim sağlayarak enfeksiyon antikor geliştirmek için belgelenmiş yeteneği ışığında özellikle önemlidir. Ayrıca, immün kompleksler fagositik alımı bağışıklık yanıtı 8 Th1/Th2 kutuplaşma etkisi dair bazı kanıtlar vardır. Burada, biz, diferansiyel IgG alt, Asn297 glukanıdır yapısının yanı sıra, yüksek verimli bir biçimde bağışıklık spesifik antijen-antikor komplekslerini oluşturmak için yeteneği neden olabilir antikor-kaynaklı fagositoz farklılıklar tespit etmek için tasarlanmış bir test tarif . Bu amaçla, 1 mikron floresan boncuk sonra klinik antikor örnekleri ile inkübe floresan antijen spesifik immün kompleksler oluşturmak, antijen ile kaplanmıştır. Bu antibody-opsonized boncuk inhibitör ve etkinleştirme de dahil olmak üzere, birden fazla FcγRs ifade monositik hücre hattı ile inkübe edilir. Testi çıkış fagositik aktivitesi, sitokin sekresyon ve FcγRs kullanım alışkanlıkları, ve bu enfeksiyon ve aşı aracılı koruma 9 hem de bu antikor-bağımlı efektör fonksiyon ayrıştırma farklılıklar için oldukça yararlı bir sistem, standart bir şekilde belirlenir.

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

Biz reseptörleri sağlayan birden fazla Fc ifade floresan antijen kaplı boncuklar ve monositik hücre hattı kullanan standart bir reseptör kullanımı ve fagositik aktivite tayinleri ve klinik örneklerde antijen-spesifik antikorların fagositik aktivitesini belirlemek için yüksek verimli bir akış sitometrik tahlil mevcut ilgi herhangi bir antijen için tekrarlanabilir moda.

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 ...

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Research

JoVE Journal

Immunology and Infection

23.3K Görüntüleme.  Ragon Institute of MGH, MIT, and Harvard. Biz reseptörleri sağlayan birden fazla Fc ifade floresan antijen kaplı boncuklar ve monositik hücre hattı kullanan standart bir reseptör kullanımı ve fagositik aktivite tayinleri ve klinik örneklerde antijen-spesifik antikorların fagositik aktivitesini belirlemek için yüksek verimli bir akış sitometrik tahlil mevcut ilgi herhangi bir antijen için tekrarlanabilir moda.

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.

Hemaglütinasyon Testi kullanarak Reaktivite ve Serum Titre Belirlenmesi

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 ...

İmmünoloji ve Enfeksiyon

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.

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 ...

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.

Oligonükleotid çiftli Floresan Mikroküreler kullanarak Kompleksi Klinik ve Çevre Örneklerinde Bakterilerin Multiplex Algılama

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.

XCELLigence Sistemi Cr51 Yayın Assay Karşılaştırılması tarafından İnsan Her2neu Özgül CD8 T hücrelerinin Optimal Sitotoksik Aktivite Belirlenmesi

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.

Ixodes scapularis Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

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.

Memeli Hücreleri ile Derneği&#39;nin Bakterilerin Canlılık belirlenmesi için Floresan Mikroskopi Yöntemleri

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.

Faj-görüntülenen Sentetik Antikor Kütüphaneler itibaren Ölçeklenebilir Yüksek Verim Seçimi

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.

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 ...

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.

Lökosit zenginleştirilmiş Kan Örneklerinde Halojenleyici Peroksidaz Faaliyet Hızlı ve Özgül Değerlendirme

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.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

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