Diese Arbeit wurde durch einen Zuschuss aus dem deutschen Bundesministerium für Bildung und Forschung gefördert (Projekt BiGRUDI [Bi ologische G efahrenlagen: R isikobewertung, u ltraschnelle D etektion und ich dentifizierung von bioterroristisch relevanten Agenzien, 13N9594]). Wir danken B. Diemar (Charité-Universitätsmedizin Berlin, Institut für Pharmakologie) für hervorragende technische Unterstützung.

1. Protokoll - IgY-Extraktion mittels PEG-Fällung Abb.. 1 zeigt eine schematische Darstellung des IgY-Extraktionsverfahren (siehe Tabelle 2). Das Protokoll wurde erstmals im Polson et al. 1980. Alle Schritte sollten unter Verwendung von Latex-Handschuhe werden. <ol><li> Die Eierschale ist sorgfältig geknackt und das Eigelb ist es, eine &quot;Eidotter Löffel&quot; übertragen, um so viel Eiweiß wie möglich zu entfernen. </li><li> Das Eigelb auf ein Filterpapier übertragen und gerollt, um restliche Eiklar zu entfernen, dann das Eigelb Haut mit einer Lanzette oder ein ähnliches Instrument (Pipettenspitze) geschnitten. Das Eigelb wird in eine 50 ml Tube gegossen und das Ei Volumen registriert ist (V1). </li><li> Zweimal das Eigelb Volumen von PBS ist mit dem Eigelb (ΣV1 + V2), danach 3,5% PEG 6000 (in Gramm, pulverisiert) des Gesamtvolumens gemischt zugegeben und gevortext, gefolgt von 10 min ins Rollen auf einer rollenden Mischer. Dieser Schritt der Extraktion trennt die Suspension in zwei Phasen. Eine Phase besteht aus &quot;Eigelb Feststoffe und fetthaltigen Substanzen&quot; (original Zitat von Polson et al. 1980) und eine wässrige Phase, die IgY und andere Proteine. </li><li> Die Röhrchen werden bei 4 ° C zentrifugiert für 20 min (10.000 rpm nach zu 13.000 xg, Heraeus Multifuge 3SR +, Festwinkelrotor). Der Überstand (V3) wird durch ein Faltenfilter gegossen und in einen neuen Schlauch. </li><li> 8,5% PEG 6000 in Gramm (berechnet nach dem neuen Volume) sind in das Röhrchen gegeben, gevortext und rollte auf einem rollenden Mischer wie in Schritt 3. </li><li> Wiederholen Sie Schritt 4 mit dem Unterschied, dass der Überstand verworfen wird. </li><li> Das Pellet wird vorsichtig in 1 ml PBS durch ein Glas-Stick und dem Vortexer aufgelöst. PBS ist ein Endvolumen von 10 ml (V4) aufgenommen. Die Lösung wird mit 12% PEG 6000 (w / v, 1,2 Gramm) gemischt und wie in Schritt 3 (Wirbel, Walzen-Mischer). </li><li> Wiederholen Sie Schritt 6 und lösen das Pellet vorsichtig in 800 ul PBS (Glas-Stick und Vortex). Warten Sie, bis die Luftblasen verschwinden und dann übertragen (Pipette) der Extrakt einer Dialyse Kapsel. Spülen Sie den Schlauch mit 400 ul PBS und fügen Sie die Lautstärke auf den Dialyse-Gerät (V5). (Zur Herstellung von Dialyse-Geräte und Membranen siehe Anhang.) </li><li> Der Extrakt wird über Nacht in 0,1% iger Kochsalzlösung (1.600 ml) dialysiert und vorsichtig durch einen Magnetrührer gerührt. Am nächsten Morgen ist die Kochsalzlösung durch PBS ersetzt und dialysiert für weitere drei Stunden. </li><li> Danach wird der IgY-Extrakt wird aus der Dialyse Kapsel mit einer Pipette gezogen und auf 2 ml Röhrchen. Das Endvolumen beträgt ca. 2 ml (V6). </li><li> Der Proteingehalt (mg / ml) der Proben wird photometrisch bei 280 nm (1:50 verdünnt mit PBS) gemessen und berechnet nach dem Lambert-Beer-Gesetz mit einem Extinktionskoeffizienten von 1,33 für IgY (siehe Abb.. 2), Abb. . 4 zeigt die Qualität der Vorbereitung (Reinheit und Erholung sind rund 80%). </li><li> Es ist ratsam, die Proben in Aliquots bei -20 ° C (nicht einfrieren die Proben bei -70 ° C). </li><li> Die Qualität der letzten Vorbereitungen wird durch einfache SDS-PAGE analysiert, wie in JoVE Protokoll beschrieben <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Appendix-vor Gebrauch der Dialyseschlauch in folgender Weise muss nach den Empfehlungen des Herstellers vorbereitet werden: <ol><li> 20 Dialyseplätze Taschen sind in Stücke von 30 cm geschnitten und da in einem 2000 ml Becherglas. </li><li> 1.750 ml einer 5 mM EDTA-Lösung versetzt. </li><li> Ein Glastrichter über dem Beutel gelegt, um sicherzustellen, dass die Beutel von der EDTA-Lösung bedeckt sind. Die Lösung wird erhitzt und für 5 min gekocht (Heizplatte). Die Lösung wird dekantiert und die Taschen sind dreimal mit destilliertem Wasser gewaschen. </li><li> Noch einmal 1.750 ml EDTA-Lösung versetzt, für 5 min gekocht und dreimal mit destilliertem Wasser wie oben. </li><li> Schließlich wird die Dialyse-Taschen sind für 10 min in destilliertem Wasser gekocht und bei 4 ° C. Nehmen Sie die Dialysebeutel durch sterilisierte Pinzette. </li></ol> 3. Repräsentative Ergebnisse <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Schematische Darstellung der IgY-Extraktion mittels Polyethylenglykol Niederschläge nach der ursprünglichen Beschreibung der Polson et al. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">Um eine größere Ansicht klicken Sie hier.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Entwicklung der Gesamt-IgY im Eidotter immunisierter Hühner in Abhängigkeit vom Alter. Da ist der wöchentliche Mittelwert der insgesamt IgY / Ei-und SD (n = 4 Legehennen). Von [Pauly et al. 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Laying Kapazität Überwachung von vier Hennen mit verschiedenen Antigenen immunisiert. Die Pfeile zeigen die Immunisierung Datum (aus [Pauly et al. 2009]). <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">Um ein größeres Bild hier klicken Ansicht.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Polyacrylamid-Gelelektrophorese einzelner IgY-Präparationen aus verschiedenen Eiern unter reduzierenden Bedingungen Lane 1 - Molekulargewichtsmarker, Spur 2 - IgY-Standard, Lane 3-5 - verschiedene IgY Proben hergestellt, wie im Protokoll beschrieben. Dies sind die letzten Vorbereitungen nach 10 des Protokolls Schritt. HC - schwere Ketten, LC - Licht chaines,? - Geringfügige Verunreinigungen entsprechend Molekulargewicht ca. 35 kDa (wahrscheinlich die C-terminale Fragment von Vitellogenin II-Vorstufe [Klimentzou et al, 2006.]). <table border="1"><tbody><tr><td>&nbsp;</td><td> Huhn 21 (Antigen A) </td><td> Huhn 22 (Antigen A) </td><td> Huhn 19 (Antigen B) </td><td> Huhn 23 (Antigen C) </td></tr><tr><td> Egg (Gesamtzahl IgY) 1, 2-Jahres-Zeitraum </td><td> 625 (38 g) </td><td> 626 (38 g) </td><td> 545 (33 g) </td><td> 608 (37 g) </td></tr><tr><td> Egg (Gesamtzahl IgY), im ersten Jahr </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 g) </td><td> 308 (17 g) </td></tr><tr><td> Egg (Gesamtzahl IgY), zweites Jahr </td><td> 280 (18 g) </td><td> 322 (21 g) </td><td> 219 (15 g) </td><td> 300 (20 g) </td></tr><tr><td> % Der max. möglichen Keim-Nummer (erstes Jahr) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Der max. möglichen Keim-Nummer (zweites Jahr) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> Anzahl der verarbeiteten Eier </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 Die Höhe der insgesamt IgY wird durch Multiplikation der Zahl der Eier durch einen mittleren Wert von 60 mg Protein pro Ei nach den Angaben in Abbildung 2 berechnet. 2 Die max. möglichen Keim-Anzahl pro Jahr liegt bei 325 nach einer Information des Züchters. Tabelle 1. Zwei Jahre Statistik der Eiablage Kapazität von Hühnern, die mit verschiedenen Antigenen immunisiert. Legen Kapazität in Prozent der maximal vier Hennen nach Immunisierung mit verschiedenen Antigenen sowie die IgY-Ergebnis innerhalb von zwei Jahren (siehe auch Abb.. 3), [Pauly et al. 2009]). <table border="1"><tbody><tr><td> Egg Nr. </td><td> Hen Nr. Legedatum </td><td> V1 [ml] Eigelb </td><td> V2 [ml] PBS </td><td> 1. PEG prec. [G] </td><td> Vol [ml] nach </td><td> 2. PEG prec. [G] </td><td> Pellet </td><td> 3. PEG prec. [G] </td><td> Pellet </td><td> Vol [ml] </td><td> Insgesamt Protein mg / ml </td><td> Korrigierte gesamten Proteins 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. und Filtration V3 </td><td> 8,5% [w / v] V3 </td><td> gelöst in ml PBS V4 </td><td> 12% [w / v] V4 </td><td> Gelöst in ml PBS V5 </td><td> Nach der Dialyse gegen PBS V6 </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> Tabelle 2. Beispielhafte Protokoll der PEG-Fällung

<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>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her, indem Landesamt für Gesundheit und Soziales Berlin (H 0069/03) eingestellt durchgeführt.

Die Wahl eines geeigneten IgY Reinigungsverfahren ist nach Skala der Reinigung (Labor oder Industrie), Wirtschaftlichkeit, Technik (Laborausstattung) und Auswirkungen auf die Umwelt (Abfallwirtschaft) beeinflusst. Verschiedene IgY Extraktionsmethoden wurden im Detail durch De Meulenaer &amp; Huyghebaert (2001, zur Überprüfung siehe auch Schade et al. 2005) überprüft. Im Allgemeinen können diese Methoden in drei Hauptgruppen eingeteilt werden: <ol><li> Niederschlag Methoden: mit Ammonium-oder Natriumsulfat, Polyethylenglykol (PEG), Caprylsäure und caragenean. </li><li> Chromatographische Methoden: Affinitätschromatographie, Ionenaustauschchromatographie, hydrophobe Interaktions-Chromatographie, thiophylic Interaktionschromatographie und Gelfiltration gereinigt. </li><li> Ultrafiltration. </li></ol> Die Reinheit eines IgY Vorbereitung kann durch eine Kombination von Methoden erhöht werden, z. B. PEG-Fällung mit Affinitätschromatographie kombiniert werden. In einigen Fällen, abhängig von der endgültigen Anwendung wird ein wässriger Extrakt der IgY ausreichend, um gute Ergebnisse (Akita &amp; Nakai 1993) zu erreichen. Nach unserer Erfahrung, arbeiteten die IgY-Probe, die wir durch PEG-Fällung erhalten sehr gut in vielen verschiedenen immunologischen Tests. Durch die Standardisierung der PEG-Fällungsverfahren eine technische Assistentin in der Lage, ungefähr 84 Eier pro Woche, was zu einem Betrag von insgesamt IgY zwischen 4 und 7 g pro Woche entspricht Prozess. Eine einfache Rechnung: Eine Legehenne ist mit einem Antigen immunisiert. Nach ein paar steigern Impfungen die Henne produziert spezifische Antikörper (Ab) mit einem Titer von 1:50.000 in einem Zeitraum von 30 Tagen. 30 Eier x 2 ml IgY-Extrakt (siehe Protokoll und Bild. 3) entspricht 60 ml insgesamt IgY, was wiederum entspricht einer Ab-Verdünnung von 3.000 l (die Kapazität einer Schaufel 10 l), die mit 3.000 l bedeutet von Ab Lösung 300 Eimer gefüllt werden kann. Also, im Hinblick auf so große Ab-Menge ist es kein Problem, eine Ab-Pool von konstanter Qualität und Spezifität zu speichern. Diese Technik reduziert die Lade / viel Variabilität sonst in polyspezifischen Ab beobachtet. Trotz der Aspekt der Quantität haben IgY Ab weitere Vorteile im Vergleich zu Säugetieren IgG: Keine Aktivierung von Säugetier-Komplement-System, keine Kreuzreaktivität mit HAMA (human anti-Maus-Antikörper), Rheumafaktoren oder menschlichen Blutgruppen-Antigene (fehlende heteroagglutinins). Ein herausragender Vorteil der IgY-Ab ist die sogenannte phylogenetische Distanz. Phylogenetische Distanz ist der Grund für die häufig beschriebenen Unterschiede zwischen den Ab Besonderheiten von Säugetieren und Geflügel, auch wenn identisch immunisiert. Neben den Unterschieden zwischen den Säugetieren und die Vogelgrippe Immunsystem selbst trägt Unterschiede in stammesgeschichtlichen Entwicklung in diesen beiden Tierklassen auf die verschiedenen Ab Besonderheiten. Mehrere Autoren haben berichtet, dass Hühner produzieren häufig Abs gegen phylogenetisch hochkonservierte Säugetieren gewonnenen Proteinen oder Peptiden effizienter als die Kaninchen (für eine Übersicht siehe Schade et a. 2005). Als Folge davon kann eine konservierte Antigen bleiben &quot;maskiert&quot;, um das Kaninchen Immunsystem und verursachen so nur einen schwachen oder eine &quot;stille&quot; Antwort. Außerdem, wenn Hühner und Kaninchen mit der gleichen Säugetieren Antigen immunisiert, sehr oft die Hühner mit einem Ab-Spezifität, die nur selten bei Kaninchen erzielt werden können reagieren. Auch der Aspekt des Tierschutzes ist wichtig, da die Ab non-invasiv werden extrahiert aus Eigelb. In Kombination mit Gene-Gun (Plasmid) Immunisierung der IgY-Produktion ist komplett nicht-invasive (Witkowski et al. 2009). Im Ergebnis ist die Herstellung von Ab in Hennen und die IgY-Extraktion mittels PEG-Fällung sehr kostengünstig und führt zu sehr spezifischen Ab mit stabilen Titer von bis zu 1:1.000.000. Aufgrund der phylogenetischen Abstand zwischen Aves und Mammalia sind Hühner in der Lage, spezifische Ak gegen hochkonservierte Säugetieren Antigene im Gegensatz zu zB Kaninchen zu produzieren. Die außergewöhnliche Höhe der Ab durch IgY-Technologie erhalten die Tür öffnet auch für den Einsatz von IgY-Ab in Human-und Veterinärmedizin zu therapeutischen / prophylaktischen Zwecken.

<ol><li> Dialyse Bag Visking, Typ 27/32, ab -14 kD, Carl Roth GmbH, Karlsruhe, Deutschland geschnitten </li><li> Falcon-Röhrchen Blue Max, 50 ml Polypropylen konisches Röhrchen, Becton Dickinson Labware, Franklin Lakes NY, USA </li><li> Faltenfilter MN 615 ¼, ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Deutschland Heraeus </li><li> Magnetrührer, Variomag Mono, H + P Labortechnik AG, Oberschleißheim, Deutschland </li><li> Micro-Dialyse-Capsule 5,0 ml, Carl Roth GmbH, Karlsruhe, Deutschland </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Deutschland </li><li> Photometer UV-160A, Shimadzu Deutschland GmbH, Duisburg, Deutschland </li><li> Polyethylenglykol 6000, Rotipuran Ph.Eur., Carl Roth GmbH, Karlsruhe, Deutschland </li><li> Premixed PBS-Puffer 10x, Roche Diagnostics GmbH, Mannheim, Deutschland </li><li> Roller Mixer SRT2, Stuart, Staffordshire, UK </li><li> Vibrofix VF2, Janke &amp; Kunkel, IKA-Labortechnik, Staufen i. Br., Deutschland </li><li> Yolk Löffel, Fackelmann (houshold Effekte), Deutschland </li></ol>

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

Hens kann durch im Impfung (Musculus pectoralis, links und rechts, Injektionsvolumen 0,5-1,0 ml) oder mit Hilfe von Gene-Gun-Plasmid-Immunisierung immunisiert werden. Abhängig von der Immunogenität des Antigens, hohe Antikörper-Titer (bis 1:100.000 - 1:1.000.000) kann nach nur einem oder 3 erreicht werden - 4 steigern Impfungen. Normalerweise legt eine Henne Eier kontinuierlich für etwa 72 Wochen, danach wird die Legeleistung sinkt. Dieses Protokoll beschreibt die Extraktion von insgesamt IgY aus Eigelb durch ein Niederschlag Verfahren (PEG. Polson et al. 1980). Das Verfahren beinhaltet zwei wichtige Schritte. Die erste ist die Entfernung von Lipiden und das zweite ist die Fällung von insgesamt IgY aus dem Überstand von step one. Nach der Dialyse gegen einen Puffer (in der Regel PBS) der IgY-Extrakt kann bei -20 ° C für mehr als ein Jahr gelagert werden. Die Reinheit des Extraktes beträgt rund 80%, variiert die Summe IgY pro Ei 40 bis 80 mg, abhängig vom Alter der Legehennen. Der gesamte Inhalt IgY steigt mit dem Alter der Henne von rund 40 mg / Ei bis zu 80 mg / Ei (über PEG-Fällung). Die Legeleistung einer Henne pro Jahr rund 325 Eier. Das bedeutet insgesamt möglichen Ernte von 20 g insgesamt IgY / Jahr bezogen auf eine mittlere IgY-Gehalt von 60 mg insgesamt IgY / Ei (siehe Tabelle 1).

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

Dieses Protokoll beschreibt insbesondere die Extraktion der Gesamt-IgY aus Eigelb mittels Polyethylenglykol Niederschlag und gibt allgemeine Informationen über IgY-Technologie.

IgY-Technologie: Extraction of Chicken Antikörper aus Eidotter durch Polyethylenglykol (PEG) Niederschlag

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 ...

igy-technology-extraction-chicken-antibodies-from-egg-yolk

Research

JoVE Journal

Immunology and Infection

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

64.3K Aufrufe.  Robert Koch-Institute. Dieses Protokoll beschreibt insbesondere die Extraktion der Gesamt-IgY aus Eigelb mittels Polyethylenglykol Niederschlag und gibt allgemeine Informationen über IgY-Technologie.

Serie: 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.

Isolierung von Lipoproteinpartikeln aus Hühnereigelb zur Untersuchung des bakteriellen Pathogens Fettsäure-Inkorporation in Membranphospholipide

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

Forschung

Biochemie

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.

Mechanische Trennung und Proteinlöslichkeit der äußeren und inneren Perivitelline-Sublayer aus Henneneiern

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.

Antikörper Bindungsspezifität für Kappa (V &amp; kgr;) Leichte Kette enthaltende Human (IgM) Antikörper: Polysialinsäure (PSA) An NCAM als Fallstudie

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

Immunologie und Infektion

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.

Standardisierung eines zytometrischen Bead-Assays auf Basis von Eigelb-Antikörpern

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.

Isolierung von Präadipozyten aus Masthähnchen-Kieferembryonen

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

Entwicklungsbiologie

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.

Reinigung und Analyse eines monoklonalen Antikörpers aus chinesischen Hamster-Ovarialzellen mit einem automatisierten Mikrobioreaktorsystem

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

Biotechnik

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.

Schnelles Antikörper-Glykoengineering in chinesischen Hamster-Eierstockzellen

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

Antikörper-Generierung: Herstellung monoklonaler Antikörper mit Hybridomen

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

Lehre

JoVE Science Education

Fortgeschrittene Biologie

Grundlagen der Immunologie

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/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

Immunprecipitation-basierte Techniken: Reinigung endogener Proteine mit Agarose-Perlen

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/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.

Eine Technik zur Aufreinigung monoklonaler Antikörper, die von Ovarialzellen des chinesischen Hamsters produziert werden

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 ...