Este trabajo fue apoyado por una subvención del Ministerio Federal Alemán de Educación e Investigación (Proyecto BiGRUDI [Bi ologische efahrenlagen G: R isikobewertung, u ltraschnelle D etektion und von I dentifizierung bioterroristisch relevanten Agenzien, 13N9594]). Agradecemos a B. Diemar (Charité-Universitätsmedizin Berlin, Institut für Pharmakologie) por su excelente asistencia técnica.

1. Protocolo - IgY-extracción por medio de la precipitación con PEG- Fig. 1 ofrece un diagrama del procedimiento de extracción de IgY (ver Tabla 2). El protocolo fue descrito por primera vez en Polson et al. 1980. Todos los pasos deben realizarse utilizando guantes de látex. <ol><li> La cáscara del huevo es cuidadosamente agrietada y la yema se transfiere a una &quot;cuchara de yema&quot; con el fin de eliminar la mayor cantidad de huevos blancos como sea posible. </li><li> La yema se transfiere a un papel de filtro y rodó a eliminar los restantes clara de huevo, la yema se corta la piel con una lanceta o un instrumento similar (pipeta). La yema se vierte en un tubo de 50 ml y el volumen del huevo está registrado (V1). </li><li> El doble del volumen de yema de huevo de PBS se mezcla con la yema de huevo (ΣV1 + V2), después de PEG 6000 al 3,5% (en gramos, pulverizada) del volumen total se agrega y se agitaron, seguido de 10 minutos rodando sobre una mesa de mezclas de laminación. Ese paso del procedimiento de extracción se separa de la suspensión en dos fases. Una fase consiste en &quot;sólidos en la yema y sustancias grasas&quot; (cita original de Polson et al. 1980) y una fase acuosa que contiene las proteínas IgY y otros. </li><li> Los tubos se centrifuga a 4 ° C durante 20 minutos (10.000 rpm a 13.000 xg de acuerdo, Heraeus Multifuge 3SR +, rotor de ángulo fijo). El sobrenadante (V3) se vierte a través de un filtro de pliegues y se transfiere a un tubo nuevo. </li><li> 8,5% de PEG 6000 en gramos (calculado de acuerdo con el nuevo volumen) se añaden al tubo, vortex y rodó por un mezclador móvil como en el paso 3. </li><li> Repita el paso 4 con la diferencia que se desecha el sobrenadante. </li><li> El pellet es cuidadosamente disuelto en 1 ml de PBS por medio de una varilla de vidrio y el vórtice. PBS se añade a un volumen final de 10 ml (V4). La solución se mezcla con PEG 6000 al 12% (w / v, 1,2 gramos) y se trata como en el paso 3 (vortex, mezclador de rodadura). </li><li> Repita el paso 6 y disolver el precipitado con cuidado en 800 l de PBS (varilla de vidrio y agitar). Espere a que las burbujas de aire a desaparecer y luego transferir (pipeta) el extracto de una cápsula de diálisis. Enjuague el tubo con 400 l de PBS y añadir el volumen de la máquina de diálisis (V5). (Para la preparación de dispositivos de diálisis y de las membranas véase el apéndice). </li><li> El extracto se dializó durante la noche en el 0,1% de solución salina (1.600 ml) y se agita suavemente por medio de un agitador magnético. A la mañana siguiente, la solución salina se sustituye por PBS y se dializó durante otras tres horas. </li><li> Posteriormente, la IgY-extracto sacado de la cápsula de diálisis por una pipeta y se transfirió a tubos de 2 ml. El volumen final es de alrededor de 2 ml (V6). </li><li> El contenido de proteína (mg / mL) de las muestras se mide fotométricamente a 280 nm (1:50 diluido en PBS) y se calcula de acuerdo con la ley de Lambert-Beer, con un coeficiente de extinción de 1,33 para el de IgY (ver fig. 2), Fig. . 4 muestra la calidad de la preparación (la pureza y la recuperación son alrededor del 80%). </li><li> Es recomendable almacenar las muestras en alícuotas a -20 ° C (no congelar las muestras a -70 ° C). </li><li> La calidad de los preparativos finales se analiza mediante una simple SDS-PAGE como se describe en el protocolo JoVe <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Apéndice antes de usar la bolsa de diálisis debe estar preparado de la siguiente manera de acuerdo a las recomendaciones del fabricante: <ol><li> 20 bolsas de diálisis se cortan en trozos de 30 cm y la da en un vaso de vidrio de 2000 ml. </li><li> 1.750 ml de una mM EDTA 5-solución se agregan. </li><li> Un embudo de vidrio se coloca por encima de las bolsas para asegurarse de que las bolsas están cubiertos por la solución de EDTA. La solución se calienta y se hierve durante 5 minutos (placa caliente). La solución se decanta y las bolsas se lavan tres veces con agua destilada. </li><li> Una vez más, 1.750 ml de solución EDTA se agregan, se hierve durante 5 minutos y se lavan tres veces con agua destilada que el anterior. </li><li> Finalmente las bolsas de diálisis se hierven durante 10 minutos en agua destilada y se almacenan a 4 ° C. Saque las bolsas de diálisis por medio de pinzas esterilizadas. </li></ol> 3. Los resultados representativos <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de IgY-extracción por medio de precipitación con polietilenglicol de acuerdo a la descripción original de Polson et al. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">Para ver una imagen más grande, haga clic aquí.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Figura 2" /> Figura 2. Desarrollo del total-IgY en la yema de huevo de gallinas inmunizadas en la dependencia de la edad. Teniendo en cuenta es la media semanal del total de IgY / huevo y el SD (n = 4 gallinas ponedoras). A partir de [Pauly et al. 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Figura 3" /> Figura 3. Layicapacidad de control ng de cuatro gallinas inmunizadas con antígenos diferentes. Las flechas indican la fecha de vacunación (de [Pauly et al. 2009]). <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">Para ver una imagen más grande, haga clic aquí.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Figura 4" /> Figura 4. Poliacrilamida-electroforesis en gel de los preparativos IgY individuales a partir de huevos diferentes condiciones reductoras Carril 1 - Marcador de peso molecular, Carril 2 - IgY-estándar, Lane 05.03 - las diferentes muestras IgY preparado como se describe en el protocolo. Estos son los últimos preparativos de acuerdo con el paso 10 del protocolo. HC - cadenas pesadas, LC - CHAINES luz? - Impurezas menores correspondiente al peso molecular alrededor de 35 kDa (probablemente el fragmento C-terminal de la vitelogenina precursor II, [Klimentzou et al, 2006.]). <table border="1"><tbody><tr><td>&nbsp;</td><td> Pollo 21 (un antígeno) </td><td> Pollo 22 (un antígeno) </td><td> Pollo 19 (B antígeno) </td><td> Pollo 23 (C antígeno) </td></tr><tr><td> El número de huevos de gallina (IgY total) 1, 2 años de período </td><td> 625 (38 g) </td><td> 626 (38 g) </td><td> 545 (33 g) </td><td> 608 (37 g) </td></tr><tr><td> Huevo número (IgY total), el primer año </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 g) </td><td> 308 (17 g) </td></tr><tr><td> Huevo número (IgY total), el segundo año </td><td> 280 (18 g) </td><td> 322 (21 g) </td><td> 219 (15 g) </td><td> 300 (20 g) </td></tr><tr><td> % Del máximo. el número de huevos posibles (primer año) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Del máximo. el número de huevos posibles (segundo año) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> número de huevos procesados </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 La cantidad de IgY total se calcula multiplicando el número de huevos por un valor medio de 60 mg de proteína por huevo de acuerdo a los datos de la Figura 2. 2 the Max. el número de huevos posible por año es de 325 de acuerdo con una información del obtentor. Tabla 1. Dos años de estadísticas de la capacidad de puesta de huevos de gallinas inmunizadas con antígenos diferentes. Acostado en la capacidad por ciento del máximo de cuatro gallinas después de la vacunación con antígenos diferentes, así como el resultado IgY-durante dos años (véase también la fig. 3), [Pauly et al. 2009]). <table border="1"><tbody><tr><td> N º de huevos. </td><td> Hen N º. Fecha de puesta </td><td> V1 [ml] yema </td><td> V2 [ml] PBS </td><td> 1. PEG prec. [G] </td><td> Vol [ml] después de </td><td> 2. PEG prec. [G] </td><td> Bolita </td><td> 3. PEG prec. [G] </td><td> Bolita </td><td> Vol [ml] </td><td> Proteínas totales mg / ml </td><td> Proteína total corregido 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. y la filtración V3 </td><td> 8,5% [w / v] V3 </td><td> disuelto en PBS ml V4 </td><td> 12% [w / v] V4 </td><td> Disuelto en PBS ml V5 </td><td> Después de la diálisis contra 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> Tabla 2. Protocolo ejemplar de PEG precipitaciones

<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>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por Landesamt für Gesundheit und Soziales Berlín (H 0069/03).

La elección de un método de purificación de IgY adecuado es influenciado por la escala de purificación (de laboratorio o industrial), la rentabilidad, la tecnología (equipos de laboratorio), y el impacto sobre el medio ambiente (gestión de residuos). Diferentes métodos de extracción de IgY se han revisado en detalle por De Meulenaer y Huyghebaert (2001, para una revisión ver también Schade et al. 2005). En general, estos métodos se pueden dividir en tres grupos principales: <ol><li> Métodos de precipitación: la participación de sulfato de amonio o de sodio, polietilenglicol (PEG), el ácido caprílico y caragenean. </li><li> Los métodos cromatográficos: cromatografía de afinidad, cromatografía de intercambio iónico, cromatografía de interacción hidrofóbica, cromatografía de interacción thiophylic, y la cromatografía de filtración en gel. </li><li> Ultrafiltración. </li></ol> La pureza de un preparado de IgY puede ser aumentado por una combinación de métodos, por ejemplo, la precipitación con PEG se puede combinar con la cromatografía de afinidad. En algunos casos, dependiendo de la aplicación final, un extracto de agua de la IgY es suficiente para lograr buenos resultados (Akita y Nakai, 1993). De acuerdo con nuestra experiencia, la IgY de la muestra se obtiene por la precipitación con PEG-funcionó muy bien en un montón de diferentes ensayos inmunológicos. Al estandarizar el método de precipitación con PEG-un asistente técnico es capaz de procesar aproximadamente 84 huevos por semana, lo que corresponde a una cantidad de IgY total entre 4 y 7 g por semana. Un simple cálculo: una gallina ponedora está inmunizado con un antígeno. Después de un impulso pocas vacunas de la gallina produce anticuerpos específicos (Ab) con un título de 1:50.000 durante un período de 30 días. 30 huevos x 2 ml de extracto de IgY (véase el protocolo y la figura. 3) corresponde a 60 ml IgY total, que a su vez corresponde a un Ab-dilución de 3.000 l (la capacidad de un cubo es de 10 l), es decir, con 3.000 l de la solución de Ab 300 cubos se pueden llenar. Por lo tanto, en vista de tan enorme cantidad de Ab-no es un problema de almacenar un Ab-piscina de calidad constante y la especificidad. Esta técnica reduce la variabilidad de carga / mucho de lo contrario observado en poliespecífico Ab. A pesar del aspecto de la cantidad, la IgY Ab tienen más ventajas en comparación con los mamíferos IgG: No activación del sistema de complemento mamífero, sin reactividad cruzada con HAMA (anticuerpos humanos anti-ratón), el factor reumatoide o los antígenos de grupo sanguíneo (falta de heteroagglutinins). Una ventaja sobresaliente de IgY-Ab es la distancia filogenética llamada. Distancia filogenética es la razón de las diferencias describe con frecuencia entre las especificidades Ab de mamíferos y aves, aunque de forma idéntica inmunizados. Además de las diferencias entre los mamíferos y el sistema inmunitario aviar sí mismos, las diferencias en el desarrollo filogenético de estas dos clases de animales contribuye a las diferentes especificidades Ab. Varios autores han reportado que los pollos a menudo producen Acs contra filogenéticamente muy conservadas proteínas o péptidos de mamíferos más eficientemente que los conejos (para revisión ver Schade et a. 2005). Como consecuencia, un antígeno conservado puede permanecer &quot;oculta&quot; para el sistema inmune de conejo, y por lo tanto la única causa una debilidad o una respuesta &quot;en silencio&quot;. Además, si los pollos y los conejos son inmunizados con el antígeno de mamíferos mismo, muy a menudo los pollos responden con un Ab-especificidad que rara vez se puede lograr en los conejos. También el aspecto de la protección de los animales es importante ya que el Ab son de forma no invasiva extraída de la yema de huevo. En combinación con (plásmido) Gene-Gun de vacunación del IgY-producción es completamente no invasivo (Witkowski et al. 2009). En conclusión, la producción de Ab en las gallinas y la extracción de IgY-por medio de PEG-precipitación es muy rentable y los resultados de Ab muy específicas con títulos estables hasta 1:1.000.000. Debido a la distancia filogenética entre Aves y Mamíferos, pollo son capaces de producir Ab específica contra los antígenos altamente conservados de mamíferos, en contraste con los conejos, por ejemplo. La extraordinaria cantidad de Ab obtenidos por la tecnología IgY-abre la puerta también para el uso de IgY-Ab en la medicina humana y veterinaria con fines terapéuticos y / o profiláctico.

<ol><li> Dializa Visking bolsa, tipo 27/32, corte -14 kD, Carl Roth GmbH, Karlsruhe, Alemania </li><li> Tubos Falcon Blue Max, 50 ml de polipropileno tubo cónico, Becton Dickinson Labware, Franklin Lakes NY, EE.UU. </li><li> Filtro plegado MN 615 ¼, ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Alemania Heraeus </li><li> Agitador magnético, Variomag Mono, H + P Labortechnik AG, Oberschleißheim, Alemania </li><li> Micro-dializa-Cápsula de 5,0 ml, Carl Roth GmbH, Karlsruhe, Alemania </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Alemania </li><li> Fotómetro UV-160A, Shimadzu Deutschland GmbH, Duisburg, Alemania </li><li> Polietilenglicol 6000, Rotipuran F. Eur., Carl Roth GmbH, Karlsruhe, Alemania </li><li> Premezclado PBS-Buffer 10x, Roche Diagnostics GmbH, Mannheim, Alemania </li><li> Roller Mixer SRT2, Stuart, Staffordshire, Reino Unido </li><li> Vibrofix VF2, Janke y Kunkel, IKA-Labortechnik, Staufen i.Br., Alemania </li><li> Cuchara de yema de huevo, Fackelmann (efectos Electricidad, gas), Alemania </li></ol>

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

Las gallinas pueden ser inmunizados a través de im vacunación (Musculus pectoral izquierda y derecha, el volumen de inyección de 0,5-1,0 ml) o por medio de la Gene-Gun plásmido de la vacunación. Depende de la inmunogenicidad de los antígenos, anticuerpos altos títulos (hasta 1:100.000 - 1:1.000.000) se puede lograr después de sólo uno o 3-4 vacunas impulso. Normalmente, la gallina de los huevos de forma continua durante aproximadamente 72 semanas, a partir de entonces la imposición de reducciones de capacidad. Este protocolo describe la extracción de IgY total de yema de huevo por medio de un procedimiento de precipitación (PEG. Polson et al. 1980). El método consiste en dos pasos importantes. El primero es la eliminación de los lípidos y el segundo es la precipitación de la IgY total del sobrenadante de un solo paso. Después de la diálisis contra un tampón (normalmente PBS) de la IgY a extraer puede ser almacenado a -20 ° C durante más de un año. La pureza del extracto es de alrededor de 80%, el total de IgY por huevo varía de 40-80 mg, dependiendo de la edad de la gallina ponedora. El incremento total de IgY contenido con la edad de la gallina de alrededor de 40 mg / huevo hasta 80 mg / huevo (en relación con PEG precipitaciones). La capacidad de colocación de una gallina por año es de alrededor de 325 huevos. Eso significa una cosecha total potencial de 20 g IgY total / año en base a un contenido de IgY media de 60 mg total de IgY de huevos / (ver Tabla 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

Este protocolo describe en particular la extracción de IgY total de yema de huevo por medio de precipitación con polietilenglicol y ofrece información general acerca de la tecnología IgY.

Tecnología IgY: Extracción de anticuerpos de pollo de yema de huevo por el polietilenglicol (PEG) Precipitación

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

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

Research

JoVE Journal

Immunology and Infection

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

64.2K vistas.  Robert Koch-Institute. Este protocolo describe en particular la extracción de IgY total de yema de huevo por medio de precipitación con polietilenglicol y ofrece información general acerca de la tecnología 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.

Reconocimiento de la transglutaminasa epidérmica por IgA y anticuerpos transglutaminasa tisular 2 en un raro caso de Rhesus Dermatitis

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

Investigación

Inmunología e Infección

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 protocolo Chelex simple para la extracción de ADN a 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.

La detección de la aspergilosis pulmonar invasiva en pacientes con neoplasia hematológica maligna, mediante el uso de la tecnología de flujo lateral

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.

La purificación y la visualización de lipopolisacárido de bacterias Gram-negativas por Hot acuosa de fenol-Extracción

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.

Propagación del virus de la influenza en embrionados de pollo Huevos

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.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

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.

Generación de anticuerpos monoclonales murino por el hibridoma Tecnología

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 método para evaluar las funciones efectoras mediadas por Fc inducidas por los anticuerpos específicos de la hemaglutinina de la gripe

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.

Generación de anticuerpos monoclonales humanos discriminativo de células B antígeno específicas raras circulan en sangre

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.

Extracción de hemocitos de larvas de Drosophila melanogaster para infección microbiana y análisis

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