Questo lavoro è stato sostenuto da una sovvenzione da parte del ministero federale tedesco dell&#39;Istruzione e della Ricerca (Progetto BiGRUDI [Bi ologische efahrenlagen G: R isikobewertung, u ltraschnelle D etektion und ho dentifizierung von bioterroristisch relevanten Agenzien, 13N9594]). Ringraziamo B. Diemar (Charité-Universitätsmedizin Berlin, Institut für Pharmakologie) per un&#39;eccellente assistenza tecnica.

1. Protocollo - IgY-estrazione con PEG-precipitazione Fig. 1 riporta lo schema del IgY estrazione procedura (cfr. tabella 2). Il protocollo è stato descritto nel Polson et al. 1980. Tutti i passaggi devono essere effettuate utilizzando guanti di lattice. <ol><li> Il guscio è incrinato con cura e il tuorlo è trasferito in un &quot;cucchiaio tuorlo&quot; al fine di rimuovere come un uovo più bianco possibile. </li><li> Il tuorlo è trasferito in una carta da filtro e laminati per rimuovere restante albume, tuorlo poi la pelle è tagliata con un bisturi o uno strumento simile (puntale). Il tuorlo viene versato in un tubo da 50 ml e il volume dell&#39;uovo è registrata (V1). </li><li> Il doppio del volume tuorlo d&#39;uovo di PBS è mescolato con l&#39;(ΣV1 + V2) tuorlo, dopo PEG 6000 del 3,5% (in grammi, polverizzati) del volume totale è aggiunto e in agitazione, seguiti da 10 minuti che rotola su un mixer di rotolamento. Questo passo della procedura di estrazione separa la sospensione in due fasi. Una fase consiste nel &quot;solidi tuorlo e sostanze grasse&quot; (citazione originale di Polson et al. 1980) e una fase acquosa contenente proteine ​​IgY e altri. </li><li> I tubi vengono centrifugati a 4 ° C per 20 min (10.000 rpm in base a 13.000 xg, Heraeus Multifuge 3SR +, rotore ad angolo fisso). Il supernatante (V3) è versato attraverso un filtro a pieghe e trasferito in una nuova provetta. </li><li> PEG 6000 8,5% in grammi (calcolato in base al nuovo volume), vengono aggiunti al tubo, in agitazione e arrotolato su un mixer a rotazione come nel punto 3. </li><li> Ripetere il punto 4 con la differenza che il supernatante viene scartato. </li><li> Il pellet viene accuratamente sciolto in 1 ml di PBS per mezzo di un bastoncino di vetro e il vortex. PBS viene aggiunto a un volume finale di 10 ml (V4). La soluzione è mescolata con 6000 PEG 12% (w / v, 1,2 grammi) e trattati come al punto 3 (vortice, mixer rotolamento). </li><li> Ripetere il passaggio 6 e sciogliere il precipitato con cura in 800 microlitri di PBS (bastone di vetro e vortex). Attendere che le bolle d&#39;aria a scomparire e poi il trasferimento (pipetta) l&#39;estratto di una capsula dialisi. Lavare il tubo con 400 microlitri di PBS e aggiungere il volume al dispositivo di dialisi (V5). (Per la preparazione di dispositivi di dialisi e le membrane vedi appendice). </li><li> L&#39;estratto è dializzato durante la notte in soluzione salina 0,1% (1.600 ml) e delicatamente mescolato per mezzo di un agitatore magnetico. La mattina seguente, la salina, è sostituito dal PBS e dializzati per altre tre ore. </li><li> Successivamente, la IgY estratto è tirato dalla capsula dialisi da una pipetta e trasferito in provette 2 ml. Il volume finale è di circa 2 ml (V6). </li><li> Il contenuto di proteine ​​(mg / mL) dei campioni è misurata fotometricamente a 280 nm (1:50 diluito con PBS) e calcolato in base alla legge di Lambert-Beer con un coefficiente di estinzione di 1,33 per IgY (vedi fig. 2), Fig. . 4 mostra la qualità della preparazione (la purezza e il recupero sono circa l&#39;80%). </li><li> Si consiglia di conservare i campioni in aliquote a -20 ° C (non congelare i campioni a -70 ° C). </li><li> La qualità della preparazione finale è analizzato da semplici SDS-PAGE, come descritto nel protocollo JOVE <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Appendice-prima di usare il sacco a dialisi deve essere preparato nel modo seguente secondo le raccomandazioni del costruttore: <ol><li> 20 sacchi dialisi vengono tagliate a pezzi di 30 cm e dato in un bicchiere di vetro 2000 ml. </li><li> 1.750 ml di 5 mm con EDTA soluzione vengono aggiunti. </li><li> Un imbuto di vetro è posto sopra i sacchetti per garantire che le borse sono coperti dalla soluzione di EDTA. La soluzione è riscaldata e bollito per 5 min (piastra calda). La soluzione è decantato e le borse sono lavati tre volte con acqua distillata. </li><li> Ancora una volta 1.750 ml con EDTA soluzione sono aggiunti, bolliti per 5 minuti e lavati tre volte con acqua distillata come sopra. </li><li> Infine le borse di dialisi vengono bolliti per 10 minuti in acqua distillata e conservato a 4 ° C. Estrarre le borse dialisi per mezzo di pinzette sterilizzate. </li></ol> 3. Rappresentante risultati <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Figura 1" /> Figura 1. Schema di IgY-estrazione mediante precipitazione polietilene glicole secondo la descrizione originale di Polson et al. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">Per visualizzare l&#39;immagine ingrandita clicca qui.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Figura 2" /> Figura 2. Sviluppo del totale-IgY nel tuorlo d&#39;uovo di gallina immunizzati in dipendenza di età. Dato è la media settimanale del totale IgY / uovo e SD (n = 4 galline ovaiole). Da [Pauly et al. 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Figura 3" /> Figura 3. Layicapacità di controllo ng di quattro galline immunizzati con antigeni diversi. Le frecce indicano la data di vaccinazione (da [Pauly et al. 2009]). <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">Per visualizzare un&#39;immagine più grande clicca qui.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Figura 4" /> Figura 4. Poliacrilammide-elettroforesi in gel di preparati IgY individuali da uova diverse in condizioni riducenti Corsia 1 - marcatore di peso molecolare, Lane 2 - IgY standard, Lane 3-5 - campioni IgY diverse preparato come descritto nel protocollo. Questi sono gli ultimi preparativi in ​​base al punto 10 del protocollo. HC - catene pesanti, LC - chaines luce,? - Impurità minore, corrispondente al peso molecolare di circa 35 kDa (probabilmente il frammento C-terminale del precursore vitellogenina II, [Klimentzou et al, 2006.]). <table border="1"><tbody><tr><td>&nbsp;</td><td> Pollo 21 (Antigen A) </td><td> Pollo 22 (Antigen A) </td><td> Pollo 19 (B Antigen) </td><td> Pollo 23 (C Antigen) </td></tr><tr><td> Numero di uova (IgY totale) 1, 2 anni di periodo </td><td> 625 (38 g) </td><td> 626 (38 g) </td><td> 545 (33 g) </td><td> 608 (37 g) </td></tr><tr><td> Numero di uova (IgY totale), primo anno </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 g) </td><td> 308 (17 g) </td></tr><tr><td> Numero di uova (IgY totale), secondo anno </td><td> 280 (18 g) </td><td> 322 (21 g) </td><td> 219 (15 g) </td><td> 300 (20 g) </td></tr><tr><td> % Di max. numero possibile di uova (primo anno) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Di max. uovo numero possibile (secondo anno) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> numero di uova trattate </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 La quantità di IgY totale viene calcolato moltiplicando il numero delle uova per un valore medio di 60 mg di proteina per uovo secondo i dati della Figura 2. 2 La max. numero possibile di uova all&#39;anno, si trova a 325 in base ad un informazione del selezionatore. Tabella 1. Statistiche Due anni di capacità di deposizione delle uova di polli immunizzati con antigeni diversi. Posa capacità in percentuale del massimo di quattro galline dopo l&#39;immunizzazione con antigeni diversi, come pure la IgY-risultato nel corso di due anni (vedi anche fig. 3), [Pauly et al. 2009]). <table border="1"><tbody><tr><td> Uovo nr. </td><td> Nr. gallina. Posa Data </td><td> V1 [ml] tuorlo </td><td> V2 [ml] di PBS </td><td> 1. PEG prec. [G] </td><td> Vol. [ml] dopo </td><td> 2. PEG prec. [G] </td><td> Pallina </td><td> 3. PEG prec. [G] </td><td> Pallina </td><td> Vol. [ml] </td><td> Proteine ​​totali mg / ml </td><td> Corretto proteine ​​totali 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. e filtrazione V3 </td><td> 8,5% [w / v] V3 </td><td> disciolti in ml V4 PBS </td><td> 12% [w / v] V4 </td><td> Sciolto in ml di PBS V5 </td><td> Dopo la dialisi contro 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> Tabella 2. Protocollo esemplare di PEG precipitazioni

<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>

Gli esperimenti sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dalla pelliccia Landesamt Gesundheit und Soziales Berlin (H 0069/03).

La scelta di un metodo adeguato di purificazione IgY è influenzata dalla scala di purificazione (laboratorio o industriale), redditività, tecnologia (attrezzature di laboratorio), e l&#39;impatto sull&#39;ambiente (gestione dei rifiuti). Vari metodi di estrazione IgY sono state esaminate in dettaglio da De Meulenaer &amp; Huyghebaert (2001, per una rassegna vedi anche Schade et al. 2005). In generale, questi metodi possono essere divisi in tre gruppi principali: <ol><li> Metodi di precipitazione: coinvolgere solfato di ammonio o di sodio, polietilenglicole (PEG), acido caprilico e caragenean. </li><li> Metodi cromatografici: cromatografia di affinità, cromatografia a scambio ionico, cromatografia di interazione idrofobica, cromatografia di interazione thiophylic, gel-filtrazione e cromatografia. </li><li> Ultrafiltrazione. </li></ol> La purezza di una preparazione IgY può essere aumentata con una combinazione di metodi, ad esempio, PEG precipitazione può essere combinato con cromatografia di affinità. In alcuni casi, a seconda dell&#39;applicazione finale, un estratto di IgY acqua è sufficiente per ottenere buoni risultati (Akita &amp; Nakai 1993). Secondo la nostra esperienza, il IgY-campione si ottiene con PEG-precipitazione ha lavorato molto bene in un sacco di diversi saggi immunologici. Uniformando i PEG-precipitazione metodo di un assistente tecnico è in grado di elaborare circa 84 uova a settimana, il che corrisponde ad un importo di IgY totale tra 4 e 7 grammi a settimana. Un semplice calcolo: una gallina ovaiola è immunizzati con un antigene. Dopo un paio di vaccinazioni aumentare la gallina produce anticorpi specifici (Ab) con un titolo di 1:50.000 durante un periodo di 30 giorni. 30 uova x 2 ml IgY estratto (vedi protocollo e fig. 3) corrisponde a 60 ml IgY totale, che a sua volta corrisponde ad un Ab-diluizione di 3.000 litri (la capacità di un secchio è di 10 l), questo significa che con 3.000 l della soluzione di Ab 300 secchi possono essere riempiti. Così, in vista di tale enorme quantità di Ab-non è un problema di memorizzare un Ab-piscina di qualità costante e specificità. Questa tecnica riduce il carico / lotto altrimenti variabilità osservata in polispecifico Ab. Nonostante l&#39;aspetto della quantità, IgY Ab hanno ulteriori vantaggi rispetto ai mammiferi IgG: Nessuna attivazione del sistema del complemento mammiferi, nessun cross-reattività con HAMA (umano anticorpo di topo anti), fattori reumatoidi o umano antigeni del gruppo sanguigno (mancanza di heteroagglutinins). Un vantaggio eccezionale di IgY-Ab è la cosiddetta distanza filogenetica. Distanza filogenetica è la ragione per le differenze spesso descritto tra le specificità Ab dei mammiferi e dei polli, anche se in modo identico immunizzati. Oltre alle differenze tra i mammiferi e il sistema immunitario aviaria se stessi, le differenze di sviluppo filogenetico in queste due classi di animali contribuisce alla specificità diverse Ab. Diversi autori hanno riportato che i polli spesso producono Abs contro filogeneticamente proteine ​​altamente conservate mammiferi o peptidi in modo più efficiente di quanto non facciano i conigli (per una rassegna vedi Schade et a. 2005). Di conseguenza, un antigene conservato può rimanere &quot;mascherato&quot; per il sistema immunitario coniglio, e quindi causare solo un debole o una risposta &quot;silenziosa&quot;. Inoltre, se polli e conigli sono immunizzati con lo stesso antigene mammiferi, molto spesso i polli rispondere con un Ab-specificità che raramente possono essere raggiunti in conigli. Anche l&#39;aspetto del benessere degli animali è importante in quanto la Ab non invasiva estratto da tuorlo d&#39;uovo. In combinazione con Gene-Gun (plasmide) l&#39;immunizzazione IgY-produzione è completamente non invasivo (Witkowski et al. 2009). In conclusione, la produzione di Ab in galline e il IgY-estrazione con PEG-precipitazione è molto conveniente e risulta in Ab altamente specifico con titoli stabile fino a 1:1.000.000. A causa della distanza filogenetica tra Aves e Mammalia, pollo sono in grado di produrre Ab specifici contro gli antigeni altamente conservato mammiferi in contrasto con i conigli per esempio. La straordinaria quantità di Ab ottenuto IgY tecnologia apre le porte anche per l&#39;utilizzo IgY-Ab nella medicina umana e veterinaria per scopi terapeutici / profilattico.

<ol><li> Dialyse Visking Bag, tipo 27/32, tagliati fuori -14 kD, Carl Roth GmbH, Karlsruhe, Germania </li><li> Provette Falcon Blue Max, da 50 ml Provetta conica in polipropilene, Becton Dickinson mediche di laboratorio, Franklin Lakes NY, USA </li><li> Piegato Filtro MN 615 ¼, ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Germania Heraeus </li><li> Agitatore magnetico, Variomag Mono, H + P Labortechnik AG, Oberschleißheim, Germania </li><li> Micro-Dialyse-Capsule 5,0 ml, Carl Roth GmbH, Karlsruhe, Germania </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Germania </li><li> Fotometro UV-160A, Shimadzu Deutschland GmbH, Duisburg, Germania </li><li> Polietilene glicole 6000, Rotipuran Ph. Eur.. Carl Roth GmbH, Karlsruhe, Germania </li><li> Premiscelato PBS-Buffer 10x, Roche Diagnostics GmbH, Mannheim, Germania </li><li> Roller Mixer SRT2, Stuart, Staffordshire, Regno Unito </li><li> Vibrofix VF2, Janke &amp; Kunkel, IKA-Labortechnik, i.Br. Staufen, Germania </li><li> Tuorlo cucchiaio, Fackelmann (effetti houshold), Germania </li></ol>

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

Galline possono essere immunizzati per mezzo di im vaccinazione (muscolo pettorale, a destra ea sinistra, volume di iniezione 0,5-1,0 ml) o per mezzo di Gene-Gun plasmide-immunizzazione. Dipende l&#39;immunogenicità dell&#39;antigene, alta anticorpi titoli (fino a 1:100.000 - 1:1.000.000) può essere raggiunto dopo solo uno o 3-4 vaccinazioni spinta. Normalmente, una gallina depone uova ininterrottamente per circa 72 settimane, dopo la diminuzione della capacità di posa. Questo protocollo descrive l&#39;estrazione di IgY totale di tuorlo d&#39;uovo per mezzo di una procedura di precipitazioni (PEG. Polson et al. 1980). Il metodo prevede due passi importanti. Il primo è la rimozione dei lipidi e il secondo è la precipitazione di IgY totale dal surnatante della fase uno. Dopo la dialisi contro un buffer (normalmente PBS) il IgY-estratto può essere conservato a -20 ° C per più di un anno. La purezza del estratto è circa l&#39;80%, il totale per IgY uovo varia da 40-80 mg, dipende dall&#39;età della gallina ovaiola. Il tenore totale di IgY aumenta con l&#39;età della gallina da circa 40 mg / uovo fino a 80 mg / uovo (PEG relative precipitazioni). La capacità di posa di una gallina per anno è di circa 325 uova. Questo significa che un raccolto totale potenziale di 20 g IgY totale / anno sulla base di un contenuto IgY media di 60 mg uovo totale IgY / (vedi tabella 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

Questo protocollo descrive in particolare l&#39;estrazione di IgY totale di tuorlo d&#39;uovo per mezzo di precipitazione polietilene glicole e fornisce informazioni generali sulla tecnologia IgY.

Tecnologia IgY: Estrazione di anticorpi pollo rosso d&#39;uovo con polietilenglicole (PEG) Precipitazioni

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 Views.  Robert Koch-Institute. Questo protocollo descrive in particolare l'estrazione di IgY totale di tuorlo d'uovo per mezzo di precipitazione polietilene glicole e fornisce informazioni generali sulla tecnologia IgY.

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

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

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

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

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

Riconoscimento della transglutaminasi epidermica da IgA e 2 anticorpi transglutaminasi tissutale in un raro caso di Rhesus Dermatite

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

Ricerca

Immunologia e infezioni

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 protocollo Chelex semplice per l&#39;estrazione del DNA da<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.

Rilevamento di aspergillosi polmonare in pazienti con neoplasie ematologiche maligne utilizzando la tecnologia a flusso laterale

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.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

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.

Influenza Virus Propagazione in embrionate di pollo Uova

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.

Generazione di ricombinante umano IgG anticorpi monoclonali da immortalizzate cellule ordinati 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.

Generazione di Murine anticorpi monoclonali da ibridomi Tecnologia

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.

Un metodo per valutare le funzioni effettrici Fc-mediata indotte dagli anticorpi specifici di Hemagglutinin di riossidazione

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.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

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.

Estrazione di emociti da larve di Drosophila melanogaster per infezione microbica e l'analisi

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