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.remotexs.ntu.edu.sg/details.php?id=1916">http:/www-jove-com.remotexs.ntu.edu.sg/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="https://www-jove-com.remotexs.ntu.edu.sg/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="https://www-jove-com.remotexs.ntu.edu.sg/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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Nanyang Technological University

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JoVE Journal

Immunology and Infection

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

64.3K 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

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Membran fosfolipidler içine bakteriyel patojen yağ asidi kuruluş çalışması için tavuk yumurtası sarısı lipoprotein parçacıklarının yalıtım

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Biyokimya

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

Dış ve İç Perivitellin Sublayerlerinin Tavuk Yumurtalarından Mekanik Ayrıştırılması ve Protein Solubilizasyonu

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...

Antibody Binding Specificity for Kappa (V&#954;) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. 
This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

We present a protocol to identify protein moieties containing antigens for human and mouse IgM antibodies with specific kappa (V&#954;) light chains. This protocol is not limited to IgM class antibodies but applies to all immunoglobulin isotypes that target their antigen with sufficiently high affinity during immunoprecipitations.

Kappa (VK) için Antikor Bağlama Özgünlük Hafif Zincir içeren İnsan (IgM) Antikorlar: Polysialic Asit (PSA) bir Vaka Çalışması olarak NCAM Bağlı

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents ...

İmmünoloji ve Enfeksiyon

Standardization of a Cytometric Bead Assay Based on Egg-Yolk Antibodies

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has gained prominence in recent decades. Each microsphere that is read by the equipment represents an analysis event of the interaction capacity between the molecules under test. Thousands of these events are read in a single assay, thus ensuring high assay accuracy and reproducibility. This methodology can also be used in the validation of new inputs, such as IgY antibodies, for the diagnosis of diseases. These antibodies are obtained through immunizing chickens with the antigen of interest and then extracting the immunoglobulin from the yolk of the animals' eggs; therefore, this is a painless and highly productive method for obtaining the antibodies. In addition to a methodology for the high-precision validation of the antibody recognition capacity of this assay, this paper also presents a method for extracting these antibodies, determining the best coupling conditions for the antibodies and latex beads, and determining the sensitivity of the test.

This protocol describes a methodology for the preparation of latex beads for assays using IgY antibodies for antigen detection.

Yumurta sarısı antikorlarına dayalı sitometrik boncuk testinin standardizasyonu

Immunoassays are important tests for the detection of numerous molecular targets. Among the methods currently available, the cytometric bead assay has ...

Isolation of Preadipocytes from Broiler Chick Embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Preadipositlerin Broiler Civciv Embriyolarından İzolasyonu

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

Gelişim Biyolojisi

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Otomatik bir Mikrobioreactor sistemi kullanılarak Çin hamster yumurtlama hücrelerinden bir monoklonal antikor arıtma ve analitik

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Biyomühendislik

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Çin Hamster Yumurtalık Hücrelerinde Hızlı Antikor Glikomühendisliği

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

Education

JoVE Science Education

İleri Biyoloji

İmmünoloji

Immunoprecipitation-Based Techniques: Purification of Endogenous Proteins Using Agarose Beads

Source: Susannah C. Shissler1, Tonya J. Webb1 
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation (IP, also known as a &#39;pull-down&#39; assay) is a widely used technique that has applications in a variety of fields. First conceived in 1984, it was refined in 1988 (1, 2). The fundamental goal of IP is purification and isolation of a specific protein using an antibody against that protein. The word &#34;immuno&#34; refers to the use of an antibody while the word &#34;precipitation&#34; refers to pulling down a specific substance from a solution. The target protein might be endogenous or recombinant. Most recombinant proteins have an epitope tag (i.e. myc or flag) attached to them to simplify subsequent purification. Typically, it is easier to optimize recombinant protein IP because the antibodies against recombinant epitope tags are very strong and effective. Antibodies against endogenous proteins have extremely variable efficacy - making it much more difficult to optimize these IPs. A necessary step after immunoprecipitation is verification of purification. The isolated protein is resolved using SDS-PAGE and subsequently probed for purity by western blots (Figure 1). An important control is the use of a different antibody during the Western blot to verify pull down of the correct protein. The combination of IP with subsequent techniques is a powerful analysis tool. The goal after purification may be characterization of the protein itself by NMR, mass spectrometry, and in vitro assays, or analysis of the protein&#39;s interacting partners (i.e. protein, DNA, RNA) (3, 4, 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10502/10502fig1.jpg" /> 
Figure 1: Overview of Immunoprecipitation Procedure. Immunoprecipitation is the isolation of a specific protein using an antibody. After production of lysate from cells, there are two major steps- pre-clearing and pull down. During pre-clearing step, the cell-lysates are pre-cleared of proteins that bind to antibodies non-specifically using an isotype control antibody. In pull down step, the target protein is pulled down using a protein-specific antibody. The isolated protein is then analyzed by Western blot. Isotype antibodies and protein specific antibodies have the same constant domain, but different antigen binding domains. A key component of this protocol is Protein A/G agarose beads that bind the constant domain of antibodies- allowing immunoprecipitation of the target protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/10502/10502fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Antibodies are the key component of an immunoprecipitation that differentiate it from other forms of protein purification (i.e. nickel affinity column purification). Antibodies are molecules made by B cells that can recognize specific protein epitopes. Antibodies have two domains: constant (Fc) and antigen binding (Fab) (Figure 1). The constant domain identifies the type of antibody and dictates function in vivo. Usually, the constant domains of antibodies used for IP are mouse, rat, or rabbit IgG. The antigen binding portion of the antibody recognizes a specific epitope of a specific protein. Antibodies can recognize epitopes on folded proteins that may not exist when the protein is denatured and vice versa. Therefore, the availability of the epitope depends on protein folding - identifying an important factor to consider when choosing antibodies and conditions for IP.
Both prokaryotic and eukaryotic systems have antibody-binding proteins. In eukaryotic systems, the purpose is immune protection from bacteria while in prokaryotic systems, the purpose is protection from the immune system. Antibody-binding proteins affect IP methodology in two ways. First, there is a necessary pre-clearing step (Figure 1) to rid the lysate of proteins that bind antibodies - thereby reducing non-specific binding in the final product. This step uses an isotype antibody that has the same constant domain as but a different antibody binding domain than your protein-specific antibody. Bacterial antibody-binding proteins are the second key component of this method. After the protein-specific antibody binds the target protein, the antibody: protein complex must be pulled down (Figure 1). Proteins A, G, and L are bacterial proteins that bind the constant domain of antibodies. While bacteria use this to subvert the immune system, researchers have co-opted this system for easy antibody purification, and it is used during both the pre-clearing and pull-down steps. These proteins have different binding affinities for different species and different constant domain subtypes - another factor to consider when choosing conditions for IP. Many companies sell Protein A/G labeled agarose beads (Figure 1), pre-made spin columns, or resins to make columns. In general, beads and spin columns are used for smaller sample sizes while resins are used for bulk purification.
In this lab exercise, we demonstrate how to purify the endogenous protein c-myc, from primary murine thymocytes, using Protein A/G Plus agarose beads based basic immunoprecipitation technique. The protocol starts from cell lysate preparation and ends with the with verification of successful protein pull down using Western blot analysis.

Immunoprecipitation Techniques: Purification of Proteins

Source: Susannah C. Shissler1, Tonya J. Webb1
1 Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201
Immunoprecipitation ...

A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

Çin Hamster Yumurtalık Hücreleri Tarafından Üretilen Monoklonal Antikorları Saflaştırmak İçin Bir Teknik

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...