Bu çalışma, Alman Federal Eğitim ve Araştırma Bakanlığı tarafından hibe tarafından desteklenmiştir (Proje BiGRUDI [Bi ologische G efahrenlagen: R isikobewertung, u ltraschnelle D etektion und I dentifizierung von bioterroristisch relevanten Agenzien, 13N9594]) . Biz mükemmel teknik yardım B. Diemar (Charité-Universitätsmedizin Berlin, Institut für Pharmakologie) teşekkür ederim.

1. Protokol - PEG-yağış yoluyla IGY çıkarma Şekil 1 IGY-çıkarma prosedürünü bir şema verir (bkz. Tablo 2). Protokol ilk Polson ve ark tarif edilmiştir. 1980. Tüm adımları lateks eldiven kullanılarak yapılmalıdır. <ol><li> Kabuğu dikkatlice çatlak ve yumurta sarısı, mümkün olduğu kadar beyaz yumurta gibi ortadan kaldırmak amacıyla &quot;sarısı kaşık&quot; aktarılır. </li><li> Yumurta sarısı, bir filtre kağıdı aktarılır ve geri kalan yumurta akı kaldırmak için atılırsa, daha sonra yumurta sarısı deri bir neşter veya benzer bir alet (pipet) ile kesilir. Yumurta sarısı, 50 ml&#39;lik bir tüp içine dökülür ve yumurta hacmi (V1) kayıtlıdır. </li><li> Iki kez PBS yumurta sarısı hacmi toplam hacminin sarısı (ΣV1 + V2) ve daha sonra% 3.5 PEG 6000 (gram toz haline getirilmiş) ile karıştırılarak eklenir ve vortekslenmiş, yuvarlanan bir karıştırıcı üzerinde yuvarlanan 10 dakika ile takip ediyor. Ekstraksiyon prosedürü bu adımı iki aşamalı olarak süspansiyon ayırır. Bir faz &quot;yumurta sarısı katıların ve yağlı maddeler&quot; (Polson ark orijinal tırnak 1980) ve IGY ve diğer proteinleri içeren bir sulu faz oluşur. </li><li> Tüpler santrifüj 4 ° C de 20 dakika (10.000 rpm 13.000 xg, Heraeus Multifuge 3SR + sabit açılı rotor göre). Süpernatant (V3) katlanmış bir filtre üzerinden dökülür ve yeni bir tüpe aktarılır. </li><li> % 8.5 gram (yeni birime göre hesaplanan) PEG 6000, tüp vortekslenmiş ve 3. adımı olarak bir haddeleme mikser atılırsa. </li><li> Süpernatantı atılır farkı ile 4. adımı yineleyin. </li><li> Pelet dikkatli bir şekilde, bir cam çubuk ve vortexer yardımıyla 1 ml PBS içinde çözülür. PBS, son hacim 10 ml (V4) eklenir. Çözüm% 12 PEG 6000 (w / v, 1.2 gram) ile karıştırılır ve adım 3 (vorteks, haddeleme mikser) gibi tedavi edilir. </li><li> 6. adımı tekrarlayın ve 800 ul PBS dikkatle pelet (cam çubuk ve vorteks) çözülür. Kaybolur ve daha sonra bir diyaliz kapsül (pipet) özü aktarmak için hava kabarcıkları için bekleyin. Tüp 400 ul PBS ile durulayın ve diyaliz cihazı (V5) sesi eklemek. (Diyaliz cihazları ve membranlar hazırlanması için eke bakınız.) </li><li> Ekstresi,% 0.1 serum fizyolojik (1.600 ml), gece boyunca diyaliz ve manyetik karıştırıcı yardımıyla hafifçe karıştırılır. Ertesi sabah, tuzlu su, PBS tarafından değiştirilir ve başka bir üç saat için diyaliz. </li><li> Daha sonra bir pipetle IGY özü diyaliz kapsül çekti ve 2ml tüpleri transfer. Son hacmi 2 ml (V6) civarındadır. </li><li> Örneklerinin protein içeriği (mg / ml), 280 nm (PBS ile seyreltilmiş 1:50) (bkz. Şekil 2) fotometrik olarak ölçülür ve IGY bir nesli için 1.33 katsayısı ile Lambert-Beer kanununa göre hesaplanır, Şekil . 4 hazırlık kalitesi (saflık ve kurtarma yaklaşık% 80) gösterir. </li><li> ° C (-70 ° C&#39;de numunelerin donma yok) -20 alikotları örnekleri saklamak için tavsiye edilir. </li><li> Son hazırlıklarını kalitesi vallahi protokolü açıklandığı gibi basit SDS-PAGE ile analiz edilir <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Ek-önce diyaliz çanta üreticisinin tavsiyelerine göre aşağıdaki şekilde hazırlanmalıdır kullanabilirsiniz: <ol><li> 20 diyaliz torbalar 30 cm parçalar halinde kesilir ve 2000 ml cam beher verilmiştir. </li><li> 5 mM EDTA çözüm 1.750 ml eklenir. </li><li> Bir cam huni, çanta çanta EDTA çözeltisi ile kaplı olduğundan emin olmak için üstüne yerleştirilir. Çözüm ısıtılır ve 5 dakika (sıcak plaka) kaynatılır. Çözüm dekante ve çanta, üç kez distile su ile yıkanır. </li><li> 1.750 ml EDTA çözüm Bir kez daha eklenir, 5 dakika süreyle kaynatılır ve yukarıdaki gibi distile su ile üç defa yıkanır. </li><li> Son olarak, diyaliz çanta, distile su içinde 10 dakika kaynatılarak ve 4 saklanır ° C Steril cımbız vasıtası ile diyaliz torbaları çıkartın. </li></ol> 3. Temsilcisi sonuçları <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Şekil 1" /> Şekil 1 Polson ark orijinal açıklamasına göre polietilen glikol yağış yoluyla IGY çıkarma şematik. 1980 <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">daha büyük bir resim görmek için buraya tıklayın.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Şekil 2" /> Şekil 2 yaş bağımlılığı aşı tavuk yumurta sarısı toplam IGY Geliştirme. Toplam IGY / yumurta ve SD (n = 4 yumurtacı tavukların) haftalık ortalama verilmiştir. [Pauly 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Şekil 3" /> Şekil 3. Layidört farklı antijen ile aşılanan tavukların ng kapasite izlenmesi. Oklar aşılama tarihi ([Pauly 2009]) gösterir. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">burada daha büyük bir resim görüntülemek için tıklayın.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Şekil 4" /> Şekil 4 azaltarak koşulları altında farklı yumurtaları tek tek IGY hazırlıkları Poliakrilamid-Gelelectrophoresis Lane 1 - Moleküler ağırlık marker, Lane 2 - IGY standardı 3-5 Lane - protokolünde açıklandığı gibi farklı IGY örnekleri hazırladı. Bu protokolün 10. adıma göre son hazırlıklarını. HC - ağır zincirleri, LC - ışık chaines,? - Yaklaşık 35 kDa (muhtemelen vitellogeninin II habercisi C-terminal parçası, [Klimentzou ve ark, 2006]) molekül ağırlığına karşılık gelen küçük kirleri. <table border="1"><tbody><tr><td>&nbsp;</td><td> Tavuk 21 (Antijen A) </td><td> Tavuk 22 (Antijen A) </td><td> Tavuk 19 (Antijen B) </td><td> Tavuk 23 (Antijen C) </td></tr><tr><td> Yumurta sayısı (toplam IGY) 1, 2 yıllık bir süre </td><td> 625 (38 gr) </td><td> 626 (38 g) </td><td> 545 (33 gr) </td><td> 608 (37 g) </td></tr><tr><td> Yumurta sayısı (toplam IGY), ilk yıl </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 gr) </td><td> 308 (17 gr) </td></tr><tr><td> Yumurta sayısı (toplam IGY), ikinci yıl </td><td> 280 (18 gr) </td><td> 322 (21 gr) </td><td> 219 (15 gr) </td><td> 300 (20 g) </td></tr><tr><td> % Max. mümkün yumurta sayısı (ilk yıl) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Max. mümkün yumurta sayısı (ikinci yıl) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> işlenmiş yumurta sayısı </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 toplam IGY miktarı ortalama değer Şekil 2 verilerine göre yumurta başına 60 mg protein, yumurta sayısı çarpılarak hesaplanır . 2 max. mümkün yumurta sayısı yılda damızlık bir bilgiye göre 325. Tablo 1. İki yıl farklı antijen ile aşılanan tavukların yumurtlama kapasitesinin istatistikleri. (Şekil ayrıca bkz 3) iki yıl boyunca farklı antijenlerin yanı sıra IGY-sonuç aşıdan sonra en fazla dört tavuk yüzde kapasite Döşeme, [Pauly 2009]) . <table border="1"><tbody><tr><td> Yumurta Nr. </td><td> Tavuk Nr. Tarihi Döşeme </td><td> V1 [ml] sarısı </td><td> V2 [ml] PBS </td><td> 1. PEG Prec. [G] </td><td> Cilt [ml] </td><td> 2. PEG Prec. [G] </td><td> Topak </td><td> 3. PEG Prec. [G] </td><td> Topak </td><td> Cilt [mL] </td><td> Total protein mg / ml </td><td> Düzeltilmiş toplam protein mg / ml </td></tr><tr><td></td><td></td><td></td><td> 2xV1 ΣV1 + V2 </td><td> % 3,5 w / v] ΣV1 + V2 </td><td> Prec. centrif. ve filtrasyon V3 </td><td> % 8,5 w / v] V3 </td><td> V4 ml PBS içinde çözünmüş </td><td> % 12 [w / v] V4 </td><td> Ml PBS V5 Çözünmüş </td><td> PBS V6 karşı diyalizden sonra </td><td> A280/ml </td><td> A280/ml: 1.33 </td></tr><tr><td> 1 </td><td> H293 / 22.10. </td><td> 15 </td><td> 30 45 </td><td> 1,58 </td><td> 31 </td><td> 2,63 </td><td> 10 </td><td> 1,2 </td><td> 1,2 </td><td> 1,7 </td><td> 35,0 </td><td> 26,3 </td></tr><tr><td> 2 </td><td> H293 / 23.10. </td><td> 13 </td><td> 26 39 </td><td> 1,37 </td><td> 25 </td><td> 2,12 </td><td> 10 </td><td> 1,2 </td><td> 1,2 </td><td> 1,7 </td><td> 28,8 </td><td> 21,6 </td></tr></tbody></table> Tablo 2 PEG yağış Örnek protokol

<ol>
	<li>Akita, E. M., Nakai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+four+purification+methods+for+the+production+of+immunoglobulins+from+eggs+laid+by+hens+immunized+with+an+enterotoxigenic+E.+coli+strain.">Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain.</a> J. Immunol. Methods. 160, 207-214 (1993).</li><li>De Meulenaer, B., Huyghebaert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+purification+of+chicken+egg+yolk+immunoglobulins:+a+review.">Isolation and purification of chicken egg yolk immunoglobulins: a review.</a> Food Agricult. Immunol. 13, 275-288 (2001).</li><li>Klimentzou, P., Paravatou-Petsotas, M., Zikos, C. h., Beck, A., Skopeliti, M., Czarnecki, J., Tsitsilonis, O., Voelter, W., Livaniou, E., Evangelatos, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+immunochemical+evaluation+of+antibodies+Y+for+the+poorly,+immunogenic+polypeptide+prothymosin+alpha.">Development and immunochemical evaluation of antibodies Y for the poorly, immunogenic polypeptide prothymosin alpha.</a> Peptides. 27, 183-193 (2006).</li><li>Pauly, D., Dorner, M., Zhang, X., Hlinak, A., Dorner, B., Schade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+laying+capacity,+immunoglobulin+Y+concentration,+and+antibody+titer+development+in+chickens+immunized+with+ricin+and+botulinum+toxins+over+a+two-year+period.">Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.</a> Poultry Science. 88, 281-290 (2009).</li><li>Polson, A., von Wechmar, M. B., van Regenmortel, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+viral+IgY+antibodies+from+yolks+of+immunized+hens.">Isolation of viral IgY antibodies from yolks of immunized hens.</a> Immunol. Commun. 9, 475-493 (1980).</li><li>Schade, R., Behn, I., Erhard, M., Hlinak, A., Staak, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chicken egg yolk antibodies, production and application : IgY-Technology. , (2001).</li><li>Schade, R., Calzado, E. G., Sarmiento, R., Chacana, P. A., Porankiewicz-Asplund, J., Terzolo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+egg+yolk+antibodies+(IgY-technology):+a+review+of+progress+in+production+and+use+in+research+and+human+and+veterinary+medicine.">Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine.</a> Altern. Lab Anim. 33, 129-154 (2005).</li><li>Witkowski, P. T., Bourquain, D. R., Hohn, O., Schade, R., Nitsche, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+gun-supported+DNA+immunisation+of+chicken+for+straightforward+production+of+poxvirus-specifiv+IgY+antibodies.">Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specifiv IgY antibodies.</a> J. Immunol. Methods. 341, 146-153 (2009).</li></ol>

Landesamt kürk Gesundheit und Soziales Berlin (H 0069/03) tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Uygun bir IGY arıtma yönteminin seçimi (laboratuar veya endüstriyel) arıtma, maliyet etkinliği, teknoloji (laboratuar ekipmanları), çevre (atık yönetimi) üzerindeki etkisi ve ölçek etkilenir. Çeşitli IGY çıkarma yöntemleri De Meulenaer &amp; Huyghebaert (2001, gözden geçirme için de Schade ve ark 2005) tarafından ayrıntılı olarak incelendi. Genel olarak, bu yöntemler üç temel gruba ayrılabilir: <ol><li> Yağış yöntemleri içeren amonyum veya sodyum sülfat, polietilenglikol (PEG), kaprilik asit ve caragenean. </li><li> Kromatografik yöntemler: afinite kromatografisi, iyon değiştirme kromatografisi, hidrofobik etkileşim kromatografi, thiophylic etkileşim kromatografisi ve jel filtrasyon kromatografisi. </li><li> Ultrafiltrasyon. </li></ol> IGY bir hazırlık saflık yöntemlerinin bir kombinasyonu ile artış olabilir; örneğin, PEG yağış afinite kromatografisi ile kombine edilebilir. Bazı durumlarda, nihai uygulama bağlı olarak, IGY su ekstraktı (Akita &amp; Nakai 1993) iyi sonuçlar elde etmek için yeterli olacaktır. Deneyimlerimize göre, PEG-yağış elde IGY örnek çok farklı immünolojik testlerin bir çok iyi çalıştı. PEG çöktürme yöntemi standartlaştırarak bir teknik asistan, 4 ve 7 gr haftada arasında toplam IGY bir miktar karşılık gelen yaklaşık 84 yumurta, haftada süreci mümkün. Basit bir hesaplama: Bir yumurta tavuğu bir antijen ile aşı. Birkaç artırmak aşılar sonra tavuk, 30 günlük bir süre boyunca 1:50,000 bir titreye sahip özel antikorlar (Ab) üretir. 30 yumurta x 2 ml IGY ekstraktı (bkz. protokolü ve Şekil 3) 3.000 l anlamına da 3.000 l Ab-seyreltme (bir kova kapasitesi 10 l) karşılık gelen 60 ml toplam IGY, karşılık gelen Ab çözüm 300 kova dolu olabilir. Yani, böyle büyük Ab-miktar, sürekli kalite ve özgünlüğü bir Ab-havuzu saklamak için hiç sorun değil. Bu teknik, aksi polyspecific Ab gözlenen şarj / çok değişkenlik azaltır. Hiçbir memeli kompleman sistemi aktivasyonu, HAMA (insan anti-fare antikor), romatoid faktörler ya da insan kan grubu antijenleri (heteroagglutinins eksikliği) ile çapraz reaktivite: miktar yönü rağmen, IGY Ab memeli IgG ile karşılaştırıldığında daha fazla avantajlara sahip olacaksınız. IGY-Ab olağanüstü bir avantaj olarak adlandırılan bir filogenetik mesafe. Filogenetik mesafe aynı aşı bile, sık sık açıklanan memeliler ve tavukların Ab özgüllükleri arasındaki farklılıkların nedeni budur. Memeli ve kuş bağışıklık sisteminin kendilerini arasındaki farklılıklara ek olarak, bu iki hayvan sınıflarına filogenetik gelişim farklılıkları, farklı Ab özgüllükleri katkıda bulunur. Bazı yazarlar, tavuk genellikle daha verimli tavşan (inceleme için Schade et a. 2005) daha filogenetik son derece korunmuş memeli protein veya peptit karşı Abs üretmek bildirdin. Bunun bir sonucu olarak, korunmuş antijen tavşan bağışıklık sistemi için &quot;maskeli&quot; kalır ve böylece sadece zayıf ya da &quot;sessiz&quot; yanıtı neden. Ayrıca, tavuk ve tavşan aynı memeli antijen ile aşı eğer çok sık tavuk nadiren tavşan elde edilebilir bir Ab-özgüllük ile yanıt. Ab, yumurta sarısı çıkarılan non-invaziv olduğundan hayvan refahı açısından önemlidir. Gen-Gun (plazmid) aşılama ile kombine IGY üretim (Witkowski ve ark 2009) tamamen non-invazif. Sonuç olarak, tavuk ve PEG-yağış yoluyla IGY çıkarma Ab üretimi çok düşük maliyetli ve istikrarlı titreleri kadar 1:1,000,000 son derece spesifik Ab sonuçlar. Kuşlar ve memeliler arasında filogenetik mesafe nedeniyle, tavuk, örneğin tavşan aksine son derece korunmuş memeli antijenlere karşı spesifik Ab üretmek mümkün. IGY teknolojisi ile elde edilen Ab olağanüstü miktarda terapötik / profilaktik amaçlı insan ve veteriner tıp IGY-Ab kullanmak için de kapıyı açar.

<ol><li> -14 KD, Carl Roth GmbH, Karlsruhe, Almanya kesti Çanta Visking Tipi 27/32, Diyaliz </li><li> Falcon tüpleri Blue Max, 50 ml Polipropilen Konik Tube, Becton Dickinson Laboratuvar Franklin Lakes NY, ABD </li><li> Katlanmış Filtre MN 615 ¼ ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Almanya Heraeus </li><li> Manyetik karıştırıcı, Variomag Mono, H + P LABORTECHNIK AG, Oberschleißheim, Almanya </li><li> Micro-Diyaliz Kapsül 5,0 ml, Carl Roth GmbH, Karlsruhe, Almanya </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Almanya </li><li> Fotometre UV-160A, Shimadzu Deutschland GmbH, Duisburg, Almanya </li><li> Polietilen Glikol 6000, Rotipuran Avrupa Farmakopesi, Carl Roth GmbH, Karlsruhe, Almanya </li><li> Premix PBS-Tampon 10x, Roche Diagnostics GmbH, Mannheim, Almanya </li><li> Rulo Mikser SRT2, Stuart, Staffordshire, İngiltere </li><li> Vibrofix VF2 Janke &amp; Kunkel, IKA-LABORTECHNIK, Staufen i.Br., Almanya </li><li> Yumurta sarısı kaşık, Fackelmann (houshold etkiler), Almanya </li></ol>

igy technology: extraction of chicken antibodies from egg yolk by polyethylene glycol (peg) precipitation

Tavuklar (Musculus pektoralis, sol ve sağ, enjeksiyon hacmi 0.5-1.0 ml) im aşılama yoluyla veya plazmid Gen-Gun-bağışıklama yoluyla aşı olabilir. Antijen immünojenite bağlı olarak, yüksek antikor titreleri (1:100,000 - 1:1,000,000) sadece bir veya 3 sonra elde edilebilir - 4 artırmak aşılar. Normalde, bir tavuk, bundan sonra döşeme kapasitesi azalır, yaklaşık 72 hafta boyunca sürekli olarak yumurta bırakır. Bu protokol sayesinde bir yağış prosedürü (PEG. Polson ve ark 1980), yumurta sarısı toplam IGY çıkarma açıklamaktadır . Bu yöntem, iki önemli adımlar içerir. Bunlardan birincisi Lipidlerin kaldırılması ve ikinci bir adım süpernatant toplam IGY yağış. IGY özü bir yıldan fazla bir tamponu (PBS normalde) karşı diyaliz sonra -20 ° C&#39;de saklanabilir. Özü saflık yaklaşık% 80, toplam IGY başına yumurta yumurta tavuğu yaşına bağlı olarak, 40-80 mg arasında değişir. 80 mg / yumurta (PEG yağış ile ilgili) yaklaşık 40 mg / yumurta tavuk yaşı ile toplam IGY içeriği artar. Yılda bir tavuk döşeme kapasitesi yaklaşık 325 yumurta. 60 mg toplam IGY / yumurta (bkz. Tablo 1) ortalama IGY içerik dayalı toplam 20 g IGY / yıl toplam potansiyel bir hasat anlamına gelir.

Watch this Scientific Journal Video about IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation at JoVE.com

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Bu protokol, polietilen glikol yağış yoluyla, özellikle yumurta sarısı toplam IGY çıkarma açıklar ve, IGY teknolojisi hakkında genel bilgi verir.

IGY Teknoloji: Yumurta Sarısı Tavuk Antikorlar çıkarımı Polietilen Glikol (PEG) Yağış

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

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Immunology and Infection

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

64.1K Görüntüleme.  Robert Koch-Institute. Bu protokol, polietilen glikol yağış yoluyla, özellikle yumurta sarısı toplam IGY çıkarma açıklar ve, IGY teknolojisi hakkında genel bilgi verir.

Video: IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

Nadir bir Case IgA ve Doku Transglutaminaz 2 Antikorlar Epidermal Transglutaminaz tanınması Rhesus Dermatit

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

İmmünoloji ve Enfeksiyon

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

DNA ekstraksiyonu için A Simple Chelex Protokolü<em&gt; Anofel</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell transplant recipients1. Detection of IPA represents a formidable diagnostic challenge and, in the absence of a 'gold standard', relies on a combination of clinical data and microbiology and histopathology where feasible. Diagnosis of IPA must conform to the European Organization for Research and Treatment of Cancer and the National Institute of Allergy and Infectious Diseases Mycology Study Group (EORTC/MSG) consensus defining "proven", "probable", and "possible" invasive fungal diseases2. Currently, no nucleic acid-based tests have been externally validated for IPA detection and so polymerase chain reaction (PCR) is not included in current EORTC/MSG diagnostic criteria.
Identification of Aspergillus in histological sections is problematic because of similarities in hyphal morphologies with other invasive fungal pathogens3, and proven identification requires isolation of the etiologic agent in pure culture. Culture-based approaches rely on the availability of biopsy samples, but these are not always accessible in sick patients, and do not always yield viable propagules for culture when obtained.
An important feature in the pathogenesis of Aspergillus is angio-invasion, a trait that provides opportunities to track the fungus immunologically using tests that detect characteristic antigenic signatures molecules in serum and bronchoalveolar lavage (BAL) fluids. This has led to the development of the Platelia enzyme immunoassay (GM-EIA) that detects Aspergillus galactomannan and a 'pan-fungal' assay (Fungitell test) that detects the conserved fungal cell wall component (1 &rarr;3)-&beta;-D-glucan, but not in the mucorales that lack this component in their cell walls1,4. Issues surrounding the accuracy of these tests1,4-6 has led to the recent development of next-generation monoclonal antibody (MAb)-based assays that detect surrogate markers of infection1,5.
Thornton5 recently described the generation of an Aspergillus-specific MAb (JF5) using hybridoma technology and its use to develop an immuno-chromatographic lateral-flow device (LFD) for the point-of-care (POC) diagnosis of IPA. A major advantage of the LFD is its ability to detect activity since MAb JF5 binds to an extracellular glycoprotein antigen that is secreted during active growth of the fungus only5. This is an important consideration when using fluids such as lung BAL for diagnosing IPA since Aspergillus spores are a common component of inhaled air. The utility of the device in diagnosing IPA has been demonstrated using an animal model of infection, where the LFD displayed improved sensitivity and specificity compared to the Platelia GM and Fungitell (1 &rarr; 3)-&beta;-D-glucan assays7.
Here, we present a simple LFD procedure to detect Aspergillus antigen in human serum and BAL fluids. Its speed and accuracy provides a novel adjunct point-of-care test for diagnosis of IPA in haematological malignancy patients.

A rapid and accurate point-of-care test for invasive pulmonary aspergillosis is presented. It takes advantage of lateral-flow technology using a specific monoclonal antibody that binds to an Aspergillus antigen secreted during pulmonary infections. The assay is compatible with serum and brochoalveolar lavage and represents a novel adjunct test for disease diagnosis.

Yanal-flow teknolojisi kullanılarak Hematolojik Malignite Hastalarda invaziv pulmoner aspergillozis Algılama

Invasive pulmonary aspergillosis (IPA) is a leading cause of morbidity and mortality in haematological malignancy patients and hematopoietic stem cell ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Sıcak Sulu-fenol Ekstraksiyon tarafından Gram-negatif bakteriler arınma ve Lipopolisakkaritlerinin Görselleştirmesi

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Embriyone Tavuk Yumurta Grip Virüsü Yayılım

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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 ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Grip Hemagglutinin özel antikorlar tarafından indüklenen Fc-aracılı efektör fonksiyonları değerlendirmek için bir yöntem

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

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 ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Hemocytes mikrobiyal enfeksiyon ve çözümleme çekme--dan Drosophila melanogaster larva

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...