1. H. Büyüme Capsulatum <ol><li> 20 dakika UV ışını ile% 10 çamaşır suyu ile temizlik kabine mantar ile çalışmak için bir biyogüvenlik kabini hazırlayın. H. maya aşamasında kalıp formu BSLIII gerektirir capsulatum, Biyogüvenlik düzeyi (BSL) II uygulamaları gerektirir. Maya formu aşağıdaki adımları kullanın. </li><li> Sonraki 5 ml PBS ile 15 ml konik tüp hazırlamak. Hasat bir koloni H. capsulatum BHI kan agar plaka [şekeri 10 g / L sistein 0,1 g / L, penisilin-streptomisin,% 1 ve koyun kırmızı kan hücreleri 50 ml / L (Colorado Serum Co, Colorado, ABD) (suşu Hc ATCC G217B ) 37 ° C&#39;de ekili veya -80 ° C&#39;de dondurulmuş maya stoku bir kısım almak ve PBS ekleyin. Vorteks şiddetle hücreleri ve oda sıcaklığında 10 dakika boyunca 1100 xg santrifüj. Pellet bozmadan süpernatantı dikkatlice çıkarın ve taze PBS 10 ml ekleyin. Bu işlemi üç kez tekrarlayın. PBS ve tranfer 50 ml konik tüp 5ml hücrelerin askıya alınması. </li><li> Toplu hücreleri bozmak için, 10 ml şırınga bir Biyogüvenlik kabini (yüz koruması kullanarak) 5 kez şarkı yoluyla maya hücre süspansiyonu 26 G1 / 2 iğne u geçmektedir. Küçük, düzgün ölçekli maya izole etmek için, oda sıcaklığında 1 dakika süreyle 55 xg&#39;de hücreler santrifüj ve sonra 1 ml kaldırmak. </li><li > 16g / L glikoz, 1g / L glutamik asit 8.4mg / L, sistin 6g / L, glutamik asit ve daha sonra 37 ° hücrelerin büyümesi ile desteklenmiş HAM F12 orta 100 ml (Gibco) içeren bir steril erlen hücre süspansiyonu ° C de 48 saat ile bir kuluçka 150rpm sallayarak. </li></ol> 2. Monoklonal Antikorlar Hibridoma Büyüme ve Arıtma <ol><li> % 10 fetal buzağı serumu,% 10 NCTC,% 1 non-esansiyel amino asitler ve% 1 Penisilin-Streptomisin içeren DMEM orta seçilen hybridomas aktarın. 37 5-7 gün için bir hücre kültürü şişesi (Becton Dickenson) hücrelerin büyümesine ° C /% 5 CO 2. </li><li> Daha sonra oda sıcaklığında 10 dakika için 1100 xg&#39;de santrifüj ve orta Peletlerin bozmadan tüm süpernatantı toplamak. </li><li> Sonra, bir protein FPLC A / G reçine kullanılarak hücre süpernatant arındırmak. </li><li> MAb konsantrasyonu sonra standart olarak IgG izotip kontrol (Şekil 1B) kullanarak bir yakalama ELISA (1) ile hesaplanabilir. </li></ol> 3 Histoplasma capsulatum İnokulum Hazırlık <ol><li> HAM F-12 orta yetişen 48 saat Histoplasma capsulatum (suşu Hc ATCC G217B) maya faz kültür atın. </li><li> 10 dakika 1100 xg&#39;de hücreleri santrifüjleyin. Süpernatantı atın ve taze PBS ekleyin. </li><li> 3.2 işlemi 3 kez tekrarlayın. </li><li> Maya hücre süspansiyonu 5 kez bir şırınga kullanarak 26G1 / 2 iğne yoluyla iletin. </li><li> Süspansiyon pelet kalan hücre kümeleri için 1 dakika için 55 xg&#39;de santrifüjleyin. Yeni bir tüp tek hücre süspansiyonu içeren süpernatant aktarın. </li><li> Hematocytometer kullanarak tek bir hücre süspansiyonu numaralandırılamıyor. </li><li> 1.25 x 10 7 maya (hayatta kalma) veya 5.0 x 10 6 (CFU, sitokinler ve histoloji) suspention &lt;50 mcL (Şekil 2) elde etmek için hücre konsantrasyonu ayarlayın. </li></ol> 4. Mablar intraperitoneal İdaresi <ol><li> Fare kuyruğu ve boynun ense (Şekil 3A) Pick up. Ense, boyun, mümkün olduğunca theears (Şekil 3B ve 3C fare işleme kişi ısırmaya başını cannotturn böylece yeterli cilt aldığınızdan emin olun) yakın olarak fare hareketsiz. </li><li> (Şekil 3B) elin avuç içi hafifçe bastırılır kuyruk withthe küçük parmak sabitleyin. </li><li> Swab, iğne ve enjekte önce kan aramak için aspire yerleştirmeden önce% 70 etanol ile enjeksiyon alanında. </li><li> 26G1 / 2 kullanın, mAb çözüm 500μg (PBS, izotip kontrol antikor veya mAb 1 mL PBS içinde seyreltilmesi, test edilmesi) içeren thelower sol veya sağ alt karın kadranda (intraperitoneal kavite) içine enjekte etmek için cilt ve abdominalmuscles piercethe konum aşağı baş hayvan, iç organlar diyafram andother (Şekil 3E) özen. </li><li> Needleto sızıntı olasılığını azaltmak çekilme öncesinde kısa bir süre bekleyin. </li><li> Bir sonraki adıma geçmeden önce en az bir iki saat bekleyin. </li></ol> 5. Fare Anestezi ve intranazal Enfeksiyonlar <ol><li> 100 mg / Kg ve 10 mg / kg, sırasıyla anestezi ketamin ve ksilizin kullanarak hazırlayın. Intraperitoneal PBS yönetim, izotip kontrol antikoru, ya da eşlik eden yazılı bir protokol açıklandığı gibi deneysel antikor gerçekleştirin. MAb enjeksiyondan sonra fare anestezi ve intranazal enfeksiyon geçmeden önce iki saat bekleyin. 100 mg / Kg ve 10 mg / Kg, sırasıyla (7) anestezi ile ketamin ve ksilizin hazırlayın.<html> </li><li> 2 saat bekleme süresi sırasında, eşlik eden yazılı bir protokol açıklandığı gibi Histoplasma capsulum inokulum hazırlamak. </li><li> 5.5, itens 5.1 göre devam edin. Toe ve refleks yokluğu doğrulamak ve anestezi çalıştı onaylamak için bir cımbız ile hayvanların tutam Anestezi sonra, bir naylon veya pamuk dize hafifçe ön dişleri fare askıya. Hayvanlar askıya almak için dizeleri bağlamak için 2 sütun standları kullanın. </li><li> Mikropipet kullanarak bir farenin solunum hızı yakından izlerken nve içine yavaşça yaklaşık 50 ul inokulum yönetmek. Fare hayvan akciğerlerine (Şekil 3F) maya birikimi kolaylaştırmak için intranazal enfeksiyondan sonra 2-5 dakika bu pozisyonda kalan izin verin. </li><li> Fare enfekte edildikten sonra, gerekirse anestezi kurtarır kadar dikkatle izlenen, sıcak bir ortam (25 ve 37 ° C arasındaki sıcaklık), bir ısı lambası kullanarak, fare korumak. Fare uyanır, yiyecek ve su ad libitum erişim ile temiz bir kafes dönün. Kafeslerde hayvan tesis döndü ve temiz bir hayvan odası (BLSI) tutulur. </li></ol> 6. Survival Çalışmaları <ol><li> , Taşipne, letarji, obtundation, kilo kaybı için, klinik olarak enfekte ve enfekte sahte hayvanların değerlendirin. Hayvanlar laboratuar üyeleri ve Hayvan Bakıcılar tarafından günlük olarak günde iki kez kontrol edilmelidir. </li><li> Fare histoplasmosis, ömrünün sonuna yakın kadar, solunum hızı hafif bir artış dışında enfeksiyon genellikle hiçbir belirgin belirtileri vardır. Ilerlemiş hastalık, genellikle 10-11 gün olur, farelerin, belirgin tachypneic hale gelir ve azalmış aktivite var . Tipik olarak hızla can çekişmekte ve sona erecek. </li><li> Sıkıntılı hayvanlar, özel bir odasında CO 2 uygun ötenazi olmalı, Merhum hayvanların günlük numaralandırılan olmalıdır . </li></ol> 7. CFU Çalışmalar, Histoloji ve Sitokinler Çalışmaları <ol><li> 15 ml konik tüpler tahsil edilecek her organ için 10 ml PBS ile hazırlayın. </li><li> Daha önce gösterildiği gibi bir subletal inuculum (5.0 x 10 6) ile 7 ve 14 gün sonra enfeksiyon hayvanlar Euthanize ve akciğer, dalak ve karaciğer hemen çıkarın. </li><li> Değerlendirilmek üzere organın bir parçasını çıkarın ve formalin gecede fiksasyonu gerçekleştirmek. Bölümleri patolojik değerlendirme için mikroskop altında incelenecektir. </li><li> 5ml PBS ile 50 ml konik tüpler hazırlayın homojenize edilecek her organ için 70 mikron hücre süzgeçler ile tepesinde. Steril 5 ml&#39;lik şırınga pistonunu kullanarak 50 ml tüpler içine organların her biri ayrı ayrı kalan kısmını ıslatarak yumuşatmak. </li><li> Sonraki seri BHI kan agar plakları (2) organ homojenatlarında (1:100, 1:1000 ve 1:5000) ve her seyreltme plaka 100μL sulandırmak. </li><li> (Roche, Almanya) üreticinin talimatlarına göre homojenatlarında proteaz inhibitörü tabletleri ekleyin. </li><li> , ° C - 14 gün ve numaralandırmak CFU kadar 37 ° plakaları inkübe edin. </li><li> 10 dakika 4000xg organ homojenatlarında Santrifüj ve organı süpernatantlar kaldırmak, whish üreticiye göre standart yöntemlerle yapılan sitokin tayinlerde kullanılan kullanılır. </li></ol> 8. H. Karşı Monoklonal Antikor Koruma Capsulatum Antikor Özgünlük ve İzotip Bağımlı. <ol><li> MAb izotip H. karşı koruma kritik capsulatum, Hsp60 için IgG1 veya IgG2a mAb ile tedavi edilen farelerde IgG2b mAb Hsp60 için bir alan fareler veya izotip kontrol mAb veya PBS (Şekil 4) kontrole göre önemli ölçüde uzun süreli sağkalım var. </li><li> Ayrıca, koruyucu mAbs alan farelerde, akciğer ve dalak CFU 7 gün sayısında anlamlı bir azalma vardı ve günde 14 (Şekil 5) akciğerlerde maya sayılar 2 ve 2,5 günlük birimlerinin azalır. </li></ol><img src="/files/ftp_upload/2532/2532fig1.jpg" alt="Şekil 1" /> Şekil 1: H. Büyüme capsulatum ve hybridomas. H. elde edilen tek bir koloni (A) Sıvı HAM F-12 kültür hazırlama capsulatum bir BHI kanlı agar plakaları üzerinde büyüdü. (B), hücre kültürü şişeleri ve protein A / G reçine kullanılarak afinite kromatografisi mAb arıtma Hibridoma büyüme. <img src="/files/ftp_upload/2532/2532fig2.jpg" alt="Şekil 2" /> Şekil 2: H. intranazal enfeksiyon capsulatum inokulum hazırlanması. H. sonra elde edilen hücre süspansiyonu şırınga ve santrifüj capsulatum agrega bozulması bir hemasitometre sayarak numaralandırılmış. Hücre konsantrasyonu 1.25 ayarlanır x 10 7 (hayatta kalma deneyler) veya 5 x 10 &lt;50 mcL 6 (CFU, sitokinler ve histoloji). upload/2532/2532fig3.jpg &quot;alt =&quot; Şekil 3 &quot;/&gt; Şekil 3: mAb yönetimi sırasında Fare taşıma. (A) fare kuyruğu tarafından aldı ve (B ve C) theears için yakın, boynun ense tutarak immobilize. (D) kuyruk küçük parmak ve avuç arasında yapılır. (E) mAb çözüm 26G1 / 2 iğne ile enjekte edilir. (F) H. capsulatum inokulum nve içine yavaşça hücre süspansiyonu pipetleme intranazal yönetilmektedir. <img src="/files/ftp_upload/2532/2532fig4.jpg" alt="Şekil 4" /> Şekil 4: Hsp60 için Mablar histoplazmozis patogenezinde değiştirebilir. IgG1 500 mg, ya da IgG2a mAbs 2 saat önce enfeksiyon önemli ölçüde uzun süreli sağkalım (kontrol grubu ile karşılaştırıldığında p &lt;0.05), ancak bir IgG2b mAb ip enjeksiyonları. <img src="/files/ftp_upload/2532/2532fig5.jpg" alt="Şekil 5" /> Şekil 5: farelerin 5x10 6 Hc maya seçilen Hsp60 mAbs veya ilgisiz mAb hayvanların tedavi mAbs IgG1 ve IgG2a mantar yükünde azalma gösterdi tedavi ip ile subletal intranazal meydan sonra CFU 7 akciğerlerde tespitler ve 14 gün. Siyah çubuklar gün 7 CFU ve beyaz çubukları temsil eden 14 gün sonrası enfeksiyon (p &lt;0.001 ve 7 gün 14 gün enfeksiyonu ** p &lt;0,001).

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

Burada sunulan protokol mAb H. olduğunu gösteriyor capsulatum deneysel fare histoplazmozis değiştirebilirsiniz. Patojenlerin hücre yüzey antijenleri Mablar bir ana ve bir patojen arasındaki etkileşimi sırasında meydana gelen karmaşık dinamiklerini değiştirebilir. Bu çalışmada, intraperitoneal olarak enjekte görünüz mAbs ölümcül histoplazmozis bir fare modeli koruma arabuluculuk kurar ve aşı geliştirilmesi için aday proteinler, H2B, M antijeni (katalaz yüzey) (3) ve Hsp60 ola...

<table border="1"><tbody><tr><th> Malzeme Adı </th><th> Şirket </th></tr><tr><td> Biyolojik güvenlik kabini (BSL2) </td><td></td></tr><tr><td> 15 ml konik tüpler </td><td> Falcon, BD </td></tr><tr><td> HAM F-12 orta </td><td> Gibco </td></tr><tr><td> 37 ° C çalkalayıcı </td><td></td></tr><tr><td> Girdap </td><td></td></tr><tr><td> 50 ml konik tüpler </td><td> Falcon, BD </td></tr><tr><td> 26G1 / 2 iğne </td><td> BD </td></tr><tr><td> 10 ml şırınga </td><td> BD </td></tr><tr><td> 250 ml şişeler </td><td></td></tr><tr><td> FPLC (Hızlı protein sıvı kromatografisi) sistemi </td><td> GE Helthcare </td></tr><tr><td> ELISA okuyucu </td><td> BioTek </td></tr><tr><td> Anestezi (ketamin ve Xylazine) </td><td></td></tr><tr><td> Sütun duruyor </td><td></td></tr><tr><td> Naylon dize </td><td></td></tr><tr><td> Isı lambası </td><td></td></tr><tr><td> 70μm hücre Strainer&#39;lar </td><td> Falcon, BD </td></tr><tr><td> BHI agar plakaları </td><td> Gibco </td></tr></tbody></table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

Bu metodoloji kullanım amacı bireyleri korumak için mantar Histoplasma capsulatum ve 2) tedavi için hedef olarak virülans faktörleri rol incelemek için neden histoplazmozis önlemek veya tedavi etmek için antikor ya da aşı ile kapasitesini ilerletmek için 1). MAb oluşturmak için, fareler aşı, bağışıklık yanıtlarını laboratuvarda geliştirilen bir katı faz ELISA sistemi kullanılarak değerlendirilir ve en iyi cevaplayıcı fareler hibridoma hücreleri ile füzyon için splenositlerin izolasyonu için seçilir. C57BL / 6 farelerin yoğun H. incelemek için kullanılan capsulatum patogenezi ve gerekli verileri elde etmek için en iyi modeli sağlamak. Enfeksiyon mAbs rolünü değerlendirmek amacıyla, fareler H. mAb ya intraperitoneal yolla capsulatum veya izotip ya intravenöz (iv), intraperitoneal (ip), ya da burun içi (in) yolları kontrolü mAb eşlemeli ve daha sonra enfekte . Bilimsel literatürde, farelerde mantar enfeksiyonları için mAbs etkinliği önceki zaman noktalarında koloni formin birim (CFU) değerlendirmeler ile birlikte, bir uç nokta olarak mortalite üzerine dayanır. En iyi insan hastalığı temsil Survival (ölüm) çalışmaları gereklidir. Böylece, bizim müdahale etkinliği yeterince sağkalım eğrileri olmadan kurulacak olmaz. Bu aşının etkinliği veya virülans için mutantlar test kurulması için de geçerlidir. Histoplazmozis, fareler genellikle birkaç saat içinde ölü enerjik olmak gidin. MAb idaresi gibi bir müdahale kapasitesi başlangıçta bir hayvan hastalığı korumak olabilir, ancak hastalığın nüks kısa CFU deneyler gerçekleştirilecek olmaz hangi. Hayatta kalma ve fungal yük testleri yanı sıra, enfeksiyon (histoloji, hücresel işe alım, sitokin yanıtları) inflamatuar yanıtları inceleyin. Farelerin hayatta kalma / ölüm deneylere zaman için, morbidite belirtileri için günde en az iki kez enfekte olan ve takip edilmektedir. Fungal yükü, histopatoloji ve sitokin yanıtları değerlendirmek için, farelerin enfeksiyondan sonra çeşitli zamanlarda ötenazi. Hayvan deneyleri Albert Einstein College of Medicine Hayvan Araştırmaları Enstitüsü&#39;nün kurallarına göre yapılır.

Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL / 6 farelerinde Hc patogenezinde çalışma ve en iyi model sağlamak için kullanılmaktadır. Biz bu mantara karşı sıvısal bağışıklığın potansiyel yararları keşfetmek ve histon H2B ve ısı şoku proteini 60kDa] üretilen çeşitli mAbs intraperitoneal uygulamadan sonra koruyucu etkinlik için test ettiğim.

Karşı Monoklonal Antikorlar Pasif Yönetim H. capsulatum Ve Diğerleri Fungal Patojenler

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

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JoVE Journal

Immunology and Infection

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

11.9K Görüntüleme.  Albert Einstein College of Medicine. C57BL / 6 farelerinde Hc patogenezinde çalışma ve en iyi model sağlamak için kullanılmaktadır. Biz bu mantara karşı sıvısal bağışıklığın potansiyel yararları keşfetmek ve histon H2B ve ısı şoku proteini 60kDa] üretilen çeşitli mAbs intraperitoneal uygulamadan sonra koruyucu etkinlik için test ettiğim.

Video: Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Tüm Influenza Kantitatif Analiz Basit Slot Blot Tahliller Evrensel Antikorlar kullanarak Viral Hemagglutinins ve nöraminidaz yazın

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

İmmünoloji ve Enfeksiyon

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Hücre içi bakteriyel patojenler ile Zebra balığı Embryolarının Enfeksiyon

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Eşodaklı Mikroskopi İzole Alveoler Makrofagların anti-fungal Hareketi değerlendirilmesi

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Ölümsüzleştirilmiştir Sıralama B hücrelerinden Rekombinant İnsan IgG Monoklonal Antikorlarının Üretimi

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Hibridoma teknolojisi ile murin monoklonal antikorlar üretilmesi

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Kanda dolaşan nadir antijen spesifik B hücreleri Discriminative insan monoklonal antikorların üretimi

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Doğal ürünler karşı monoklonal antikorların üretimi

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic öldürme tahlil bakteriyel patojenlere karşı immünolojik yanıt değerlendirmek için

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...