Ce travail a été soutenu par une subvention du ministère fédéral allemand de l&#39;Education et la Recherche (Projet BiGRUDI [Bi ologische G efahrenlagen: R isikobewertung, u ltraschnelle D etektion und von j&#39;ai dentifizierung bioterroristisch relevanten Agenzien, 13N9594]). Nous remercions B. Diemar (Charité-Universitätsmedizin Berlin, Institut für Pharmakologie) pour l&#39;assistance technique excellente.

1. Protocole - IgY extraction au moyen de PEG-précipitation Fig. 1 donne le schéma de la procédure d&#39;IgY-extraction (voir tableau 2). Le protocole a été décrit pour la première à Polson et al. 1980. Toutes les mesures doivent être effectuées en utilisant des gants en latex. <ol><li> La coquille est soigneusement fissuré et le jaune est transférée à une &quot;cuillère jaune&quot; afin d&#39;éliminer autant que l&#39;œuf blanc que possible. </li><li> Le jaune est transféré à un papier filtre et roulé pour enlever les blancs d&#39;œufs, puis la peau jaune est coupé avec une lancette ou un instrument similaire (pipette). Le jaune est versé dans un tube de 50 ml et le volume d&#39;oeuf est enregistré (V1). </li><li> Deux fois le volume de PBS jaune d&#39;œuf est mélangé avec le jaune (ΣV1 + V2), puis de 3,5% de PEG 6000 (en gramme, pulvérisé) du volume total est ajouté et vortexés, suivies de 10 min roulant sur une table de mixage roulant. Cette étape de la procédure d&#39;extraction sépare la suspension en deux phases. Une phase se compose de «solides jaune et corps gras» (citation originale de Polson et al. 1980) et une phase aqueuse contenant des protéines IgY et autres. </li><li> Les tubes sont centrifugés à 4 ° C pendant 20 min (10.000 rpm en fonction de 13 000 xg, Heraeus Multifuge 3SR +, rotor à angle fixe). Le surnageant (V3) est versé à travers un filtre plissé et transféré dans un nouveau tube. </li><li> PEG 6000 de 8,5% en gramme (calculés selon le nouveau volume) sont ajoutés au tube, vortex et roulé sur une table de mixage rouler comme à l&#39;étape 3. </li><li> Répétez l&#39;étape 4 à la différence que le surnageant est éliminé. </li><li> Le culot est soigneusement dissous dans 1 ml de PBS à l&#39;aide d&#39;un bâton de verre et le Vortexer. PBS est ajouté à un volume final de 10 ml (V4). La solution est mélangée avec le PEG 6000 12% (p / v, 1,2 gramme) et traitée comme dans l&#39;étape 3 (vortex, mélangeur de roulement). </li><li> Répétez l&#39;étape 6 et dissoudre l&#39;extrait soigneusement dans 800 ul de PBS (bâton de verre et de vortex). Attendre que les bulles d&#39;air disparaissent, puis transfert (pipette) de l&#39;extrait d&#39;une capsule de dialyse. Rincer le tube avec 400 ul de PBS et ajouter du volume à l&#39;appareil de dialyse (V5). (Pour la préparation d&#39;appareils de dialyse et les membranes voir annexe). </li><li> L&#39;extrait est dialysé pendant la nuit dans une solution saline à 0,1% (1600 ml) et agite doucement à l&#39;aide d&#39;un agitateur magnétique. Le lendemain matin, la saline est remplacé par du PBS et dialysée pendant encore trois heures. </li><li> Par la suite, l&#39;AGI-extrait est tiré de la capsule de dialyse par une pipette et transférées dans des tubes de 2ml. Le volume final est d&#39;environ 2 ml (V6). </li><li> La teneur en protéines (mg / mL) des échantillons est mesurée par photométrie à 280 nm (1:50 diluée avec du PBS) et calculé conformément à la loi de Lambert-Beer avec un coefficient d&#39;extinction de 1,33 pour IgY (voir Fig. 2), figure . 4 montre la qualité de la préparation (pureté et de récupération sont environ 80%). </li><li> Il est conseillé de stocker les échantillons dans des aliquotes à -20 ° C (ne pas congeler les échantillons à -70 ° C). </li><li> La qualité de la préparation finale est analysé par SDS-PAGE simples tel que décrit dans le protocole JoVE <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Annexe avant d&#39;utiliser le sac de dialyse doit être préparé de la manière suivante selon les recommandations du fabricant: <ol><li> 20 sacs de dialyse sont découpés en morceaux de 30 cm et donnée dans un bécher de 2000 ml en verre. </li><li> 1750 ml d&#39;une 5 mM EDTA solution sont ajoutés. </li><li> Un entonnoir en verre est placée au-dessus des sacs pour s&#39;assurer que les sacs sont couverts par la solution d&#39;EDTA. La solution est chauffée et bouillir pendant 5 min (plaque chauffante). La solution est décantée et les sacs sont lavées trois fois avec de l&#39;eau distillée. </li><li> Encore une fois 1750 ml d&#39;EDTA-solution sont ajoutés, bouillir pendant 5 min et lavées trois fois avec de l&#39;eau distillée comme ci-dessus. </li><li> Enfin, les sacs de dialyse sont bouillis pendant 10 min dans de l&#39;eau distillée et conservés à 4 ° C. Sortez les sacs de dialyse à l&#39;aide d&#39;une pince à épiler stérilisée. </li></ol> 3. Les résultats représentatifs <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Figure 1" /> Figure 1. Schéma de principe d&#39;IgY-extraction au moyen de la précipitation de polyéthylène glycol selon la description originale de Polson et al. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">Pour afficher une image plus grande cliquez ici.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Figure 2" /> Figure 2. Développement du total-IgY de jaune d&#39;oeuf de poules immunisées dans la dépendance de l&#39;âge. Compte tenu de la moyenne hebdomadaire du total des IgY / œuf et SD (n = 4 poules pondeuses). De [Pauly et al. 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Figure 3" /> Figure 3. Layicapacité de suivi de quatre poules ng immunisées avec des antigènes différents. Les flèches indiquent la date de vaccination (d&#39;après [Pauly et al. 2009]). <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">Pour afficher une image plus grande cliquez ici.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Figure 4" /> Figure 4. Polyacrylamide-électrophorèse en gel de préparations IgY individuels à partir d&#39;œufs différents dans des conditions réductrices Voie 1 - marqueur de poids moléculaire, la voie 2 - IgY standard, Lane 3-5 - échantillons différents IgY préparé comme décrit dans le protocole. Ce sont les derniers préparatifs en fonction de l&#39;étape 10 du protocole. HC - chaînes lourdes, LC - chaines lumière,? - Impuretés mineures correspondant à poids moléculaire d&#39;environ 35 kDa (probablement le fragment C-terminal de la vitellogénine II précurseur, [Klimentzou et al, 2006.]). <table border="1"><tbody><tr><td>&nbsp;</td><td> Poulet 21 (antigène A) </td><td> Poulet 22 (antigène A) </td><td> Poulet 19 (B Antigen) </td><td> Poulet 23 (C Antigen) </td></tr><tr><td> Nombre d&#39;œufs (IgY total) 1, 2 ans la période </td><td> 625 (38 g) </td><td> 626 (38 g) </td><td> 545 (33 g) </td><td> 608 (37 g) </td></tr><tr><td> Nombre d&#39;œufs (IgY total), la première année </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 g) </td><td> 308 (17 g) </td></tr><tr><td> Nombre d&#39;œufs (IgY total), deuxième année </td><td> 280 (18 g) </td><td> 322 (21 g) </td><td> 219 (15 g) </td><td> 300 (20 g) </td></tr><tr><td> % Du max. nombre d&#39;œufs possible (première année) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Du max. nombre d&#39;œufs possible (deuxième année) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> nombre d&#39;œufs transformés </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 Le montant de IgY total est calculé en multipliant le nombre d&#39;oeufs par une valeur moyenne de 60 mg de protéine par oeuf, selon les données de la figure 2. Les deux max. nombre d&#39;œufs possibles par an est de 325 selon une information de l&#39;éleveur. Tableau 1. Deux statistiques des années de la capacité de ponte des poules immunisées avec des antigènes différents. Pose des capacités en pour cent du maximum de quatre poules après immunisation avec des antigènes différents ainsi que l&#39;AGI-résultat pendant deux ans (voir aussi Fig. 3), [Pauly et al. 2009]). <table border="1"><tbody><tr><td> Egg Nr. </td><td> Nr. poule. Date de ponte </td><td> V1 [ml] jaune </td><td> V2 [ml] de PBS </td><td> 1. PEG prec. [G] </td><td> Vol [ml] après </td><td> 2. PEG prec. [G] </td><td> Boulette </td><td> 3. PEG prec. [G] </td><td> Boulette </td><td> Vol [ml] </td><td> Protéines totales mg / ml </td><td> Corrigé des protéines totales 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. CENTRIFUGE. filtration et V3 </td><td> 8,5% [w / v] V3 </td><td> dissous dans du PBS ml V4 </td><td> 12% [w / v] V4 </td><td> Dissous dans ml de PBS V5 </td><td> Après dialyse contre du 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> Tableau 2. Protocole exemples de PEG précipitations

<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>

Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements stipulés par Landesamt für Gesundheit und Soziales de Berlin (H 0069/03).

Le choix d&#39;une méthode de purification IgY approprié est influencée par l&#39;échelle de la purification (laboratoire ou industrielle), la rentabilité, de la technologie (matériel de laboratoire), et l&#39;impact sur l&#39;environnement (gestion des déchets). Diverses méthodes d&#39;extraction ont été IgY examiné en détail par De Meulenaer &amp; Huyghebaert (2001, pour revue, voir aussi Schade et al. 2005). En général, ces méthodes peuvent être divisés en trois groupes principaux: <ol><li> Méthodes de précipitation: l&#39;implication de sulfate d&#39;ammonium ou de sodium, polyéthylène glycol (PEG), l&#39;acide caprylique et caragenean. </li><li> Les méthodes chromatographiques: chromatographie d&#39;affinité, chromatographie échangeuse d&#39;ions, chromatographie d&#39;interaction hydrophobe, la chromatographie d&#39;interaction thiophylic, et de filtration sur gel de chromatographie. </li><li> L&#39;ultrafiltration. </li></ol> La pureté d&#39;une préparation IgY peut être augmentée par une combinaison de méthodes, par exemple, le PEG précipitations peuvent être combinés avec la chromatographie d&#39;affinité. Dans certains cas, selon l&#39;application finale, un extrait d&#39;eau de IgY est suffisante pour obtenir de bons résultats (Akita &amp; Nakai, 1993). Selon notre expérience, l&#39;AGI-échantillon, nous obtenons par PEG-précipitation a très bien fonctionné dans beaucoup de différents dosages immunologiques. En standardisant la méthode de précipitation PEG-un assistant technique est capable de traiter environ 84 œufs par semaine, ce qui correspond à un montant de IgY totales entre 4 et 7 g par semaine. Un simple calcul: une poule pondeuse est immunisé avec un antigène. Après une vaccination stimuler la poule quelques produit des anticorps spécifiques (Ab) avec un titre de 1:50,000 pendant une période de 30 jours. 30 œufs x 2 ml d&#39;extrait IgY (voir protocole et Fig. 3) correspond à 60 ml IgY total, qui à son tour correspond à une dilution de Ab-3000 l (la capacité d&#39;un seau est de 10 l), ce qui signifie avec 3.000 l de la solution AB 300 seaux peuvent être remplies. Ainsi, en vue d&#39;une telle quantité énorme Ab-il pas un problème pour stocker une Ab-bassin de qualité constante et de spécificité. Cette technique réduit la variabilité de charge / lot contraire observé dans polyspécifique Ab. Malgré l&#39;aspect quantitatif, IgY Ab ont d&#39;autres avantages par rapport aux IgG des mammifères: Pas d&#39;activation du système du complément chez les mammifères, aucune réactivité croisée avec HAMA (anticorps humain anti-souris), les facteurs rhumatoïdes ou humaine antigènes de groupe sanguin (manque de heteroagglutinins). Un avantage remarquable de IgY-Ab est la distance phylogénétique dits. Distance phylogénétique est la raison pour laquelle les différences entre les décrit souvent les spécificités Ab des mammifères et des poules, même lorsque l&#39;identique vaccinés. En plus de différences entre les mammifères et le système immunitaire aviaire eux-mêmes, les différences dans le développement phylogénétique de ces deux classes d&#39;animaux contribue aux spécificités différentes Ab. Plusieurs auteurs ont rapporté que les poulets produisent souvent Abs contre phylogénétiquement très conservées des protéines de mammifères ou des peptides plus efficacement que ne le lapin (pour revue, voir Schade et a. 2005). En conséquence, un antigène conservé peut rester &quot;masqués&quot; pour le système de lapin immunitaire, et provoquer ainsi qu&#39;une faiblesse ou un &quot;silence&quot; de réponse. Par ailleurs, si les poulets et les lapins sont immunisés avec le même antigène de mammifères, très souvent les poulets répondre avec un Ab-spécificité qui peut rarement être obtenue chez le lapin. Aussi l&#39;aspect de bien-être animal est important puisque les Ab sont non invasive extrait de jaune d&#39;oeuf. En combinaison avec Gene-Gun (plasmide) vaccination de l&#39;IgY-production est totalement non-invasive (Witkowski et al. 2009). En conclusion, la production d&#39;Ab dans les poules et les IgY extraction au moyen de PEG-précipitation est très rentable et les résultats dans Ab très spécifique avec des titres stables jusqu&#39;à 1:1,000,000. En raison de la distance phylogénétique entre Aves et Mammalia, de poulet sont capables de produire Ab spécifiques contre les antigènes hautement conservée chez les mammifères, contrairement aux lapins par exemple. La quantité extraordinaire d&#39;Ab obtenus par IgY technologie ouvre la porte aussi à l&#39;aide IgY-Ab dans la médecine humaine et vétérinaire à des fins thérapeutiques / prophylactiques.

<ol><li> Dialyse Visking sac, de type 27/32, coupé -14 kD, Carl Roth GmbH, Karlsruhe, Allemagne </li><li> Tubes Falcon Blue Max, 50 ml en polypropylène Tube conique, Becton Dickinson Labware, Franklin Lakes NY, USA </li><li> Filtre plissé MN 615 ¼, ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Allemagne Heraeus </li><li> Agitateur magnétique, Variomag Mono, H + P Labortechnik AG, Oberschleißheim, Allemagne </li><li> Micro-Dialyse-Capsule 5,0 ml, Carl Roth GmbH, Karlsruhe, Allemagne </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Allemagne </li><li> Photomètre UV-160A, Shimadzu Deutschland GmbH, Duisburg, Allemagne </li><li> Le polyéthylène glycol 6000, Rotipuran Ph. Eur., Carl Roth GmbH, Karlsruhe, Allemagne </li><li> Prémélangé PBS-Buffer 10x, Roche Diagnostics GmbH, Mannheim, Allemagne </li><li> Rouleau Mixer SRT2, Stuart, dans le Staffordshire, Royaume-Uni </li><li> Vibrofix VF2, Janke &amp; Kunkel, IKA-Labortechnik, Staufen-en-Brisgau, Allemagne </li><li> Cuillère Jaune, Fackelmann (effets houshold), Allemagne </li></ol>

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

Les poules peuvent être immunisés par la vaccination au moyen d&#39;im (Musculus pectoralis, gauche et droite, le volume d&#39;injection 0,5-1,0 ml) ou par l&#39;intermédiaire de Gene-Gun plasmide-immunisation. Charge sur l&#39;immunogénicité de l&#39;antigène, d&#39;anticorps de haute-titres (jusqu&#39;à 1:100.000 - 1:1,000,000) peuvent être obtenus après seulement une ou 3 - 4 immunisations stimuler. Normalement, une poule pond des oeufs en continu pendant environ 72 semaines, par la suite la capacité diminue de pose. Ce protocole décrit l&#39;extraction des IgY totale de jaune d&#39;œuf par le biais d&#39;une procédure de précipitations (PEG. Polson et al. 1980). La méthode comporte deux étapes importantes. Le premier est l&#39;élimination des lipides et la seconde est la précipitation de l&#39;IgY totale à partir du surnageant de la première étape. Après dialyse contre un tampon (PBS normalement) l&#39;AGI-extrait peut être conservé à -20 ° C pendant plus d&#39;un an. La pureté de l&#39;extrait est d&#39;environ 80%, le total des IgY par oeuf varie de 40-80 mg, dépendant de l&#39;âge de la poule pondeuse. Les augmentations de la teneur totale en IgY avec l&#39;âge de la poule d&#39;environ 40 mg / oeuf jusqu&#39;à 80 mg / oeuf (concernant le PEG précipitations). La capacité de ponte d&#39;une poule par an est d&#39;environ 325 œufs. Cela signifie une récolte totale potentielle de 20 g au total IgY / an basé sur un contenu IgY moyenne de 60 mg d&#39;œuf IgY / total (voir tableau 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

Ce protocole décrit notamment l&#39;extraction de IgY totales du jaune d&#39;oeuf par précipitation de polyéthylène glycol et donne des informations générales sur la technologie IgY.

Technologie IgY: Extraction d&#39;anticorps de jaune d&#39;œuf de poulet par polyéthylène glycol (PEG) Précipitations

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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Research

JoVE Journal

Immunology and Infection

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

64.1K vues.  Robert Koch-Institute. Ce protocole décrit notamment l'extraction de IgY totales du jaune d'oeuf par précipitation de polyéthylène glycol et donne des informations générales sur la technologie IgY.

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

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

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

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

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

La reconnaissance de la transglutaminase épidermique par les IgA et Tissue Transglutaminase 2 anticorps dans un cas rare de Rhésus Dermatite

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

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Immunologie et infection

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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

Un protocole Chelex simple pour l&#39;extraction d&#39;ADN à partir de<em&gt; Anopheles</em&gt; Spp.

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

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

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

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

Détection de l&#39;aspergillose pulmonaire invasive chez les patients hémopathie maligne en utilisant la technologie de flux latéral-

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

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

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

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

Purification et visualisation de lipopolysaccharide de bactéries à Gram négatif par Hot-phénol aqueux d&#39;extraction

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

Influenza Virus Propagation in Embryonated Chicken Eggs

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

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

Virus de la grippe Propagation dans embryonnés de poulet Oeufs

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

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

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

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

Génération de Recombinant Human IgG anticorps monoclonaux à partir de cellules immortalisées Classé B

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

Generation of Murine Monoclonal Antibodies by Hybridoma Technology

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

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

La génération d&#39;anticorps monoclonaux murins par Hybridoma Technology

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

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

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

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

Une méthode pour évaluer les fonctions effectrices induite par le Fc induites par les anticorps spécifiques d’hémagglutinine de grippe

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

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

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

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

Génération de discriminatif anticorps monoclonaux humains de cellules rares spécifiques de l’antigène B circulant dans le sang

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

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

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

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

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

Extraction des hémocytes de larves de drosophile d’Infection microbienne et d’analyse

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