Este trabalho foi financiado por uma doação do Ministério Federal Alemão de Educação e Pesquisa (Projeto BiGRUDI [Bi ologische G efahrenlagen: R isikobewertung, u ltraschnelle D etektion und eu dentifizierung von bioterroristisch relevanten Agenzien, 13N9594]). Agradecemos a B. Diemar (Charité-Universitätsmedizin Berlim, Institut für Pharmakologie) para assistência técnica excelente.

1. Protocolo - IgY-extração por meio de PEG-precipitação Fig. 1 apresenta o esquema do processo de extração de IgY (ver Tabela 2). O protocolo foi descrita pela primeira vez em Polson et al. 1980. Todas as etapas devem ser executadas usando luvas de látex. <ol><li> A casca é cuidadosamente rachada ea gema é transferido para uma &quot;colher de gema&quot;, a fim de remover o ovo muito branco quanto possível. </li><li> A gema é transferido para um papel de filtro e rolou para remover restantes clara de ovo, a gema, em seguida, a pele é cortada com uma lanceta ou um instrumento similar (pipeta ponta). A gema é colocada em um tubo de 50 ml eo volume de ovos está registrado (V1). </li><li> Duas vezes o volume de gema de ovo PBS é misturado com o (ΣV1 + V2) gema, PEG 6000 depois de 3,5% (em grama, pulverizado) do volume total é adicionado e agitadas, seguido por 10 min que rola em um misturador de rolamento. Essa etapa do procedimento de extração separa a suspensão em duas fases. Uma fase consiste em &quot;sólidos gema e substâncias gordas&quot; (citação original de Polson et al. 1980) e uma fase aquosa contendo proteínas IgY e outros. </li><li> Os tubos são centrifugados a 4 ° C por 20 min (10.000 rpm de acordo com a 13000 xg Heraeus Multifuge 3SR +, rotor ângulo fixo). O sobrenadante (V3) é filtrado através de um filtro dobrado e transferidos para um novo tubo. </li><li> PEG 6000 8,5% em grama (calculado de acordo com o novo volume) são adicionados ao tubo, agitadas e rolou em um misturador evolutivo como no passo 3. </li><li> Repita o passo 4 com a diferença que o sobrenadante é descartado. </li><li> O pellet é cuidadosamente dissolvidos em 1 ml de PBS, por meio de um bastão de vidro eo vortexer. PBS é adicionado a um volume final de 10 ml (V4). A solução é misturada com PEG 6000 12% (w / v, 1,2 grama) e tratados como no passo 3 (vortex, rolando). </li><li> Repita a etapa 6 e dissolver o sedimento cuidadosamente em 800 mL de PBS (vidro vara e vortex). Espere que as bolhas de ar para desaparecer e em seguida, transferir (pipeta) o extrato de uma cápsula de diálise. Lavar o tubo com 400 mL PBS e adicionar o volume para o dispositivo de diálise (V5). (Para a preparação de dispositivos de diálise e membranas ver anexo). </li><li> O extrato é dialisada durante a noite em solução salina 0,1% (1.600 ml) e agitado suavemente por meio de um agitador magnético. Na manhã seguinte, a solução salina é substituído por PBS e dialisado por mais três horas. </li><li> Posteriormente, o extrato de IgY é retirado da cápsula de diálise através de uma pipeta e transferidas para tubos de 2ml. O volume final é de cerca de 2 ml (V6). </li><li> O teor de proteína (mg / mL) das amostras é medida por fotometria em 280 nm (1:50 diluído com PBS) e calculado de acordo com a lei de Lambert-Beer, com um coeficiente de extinção de 1,33 para IgY (ver fig. 2), Fig. . 4 mostra a qualidade da preparação (pureza e recuperação são cerca de 80%). </li><li> É aconselhável para armazenar as amostras em alíquotas a -20 ° C (não congelar as amostras a -70 ° C). </li><li> A qualidade dos preparativos finais é analisada por SDS-PAGE simples como descrito no protocolo JOVE <a href="http:/www.jove.com/details.php?id=1916">http:/www.jove.com/details.php?id=1916</a> </li></ol> 2. Apêndice-antes de usar o saco de diálise deve ser preparado da seguinte maneira de acordo com as recomendações do fabricante: <ol><li> 20 sacos de diálise são cortadas em pedaços de 30 cm e dado num copo de vidro 2000 ml. </li><li> 1750 ml de 5 mM EDTA-solução são adicionados. </li><li> Um funil de vidro é colocado acima dos sacos para garantir que os sacos são cobertos pela solução de EDTA. A solução é aquecida e fervida por 5 min (placa quente). A solução é decantado e os sacos são lavadas três vezes com água destilada. </li><li> Mais uma vez 1750 ml EDTA-solução são adicionados, fervido por 5 min e lavadas três vezes com água destilada como acima. </li><li> Finalmente os sacos de diálise são fervidos por 10 min em água destilada e armazenado a 4 ° C. Tirar os sacos de diálise por meio de pinças esterilizadas. </li></ol> 3. Resultados representativos <img src="/files/ftp_upload/3084/3084fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama esquemático de IgY-extração por meio de precipitação de polietileno glicol de acordo com a descrição original de Polson et al. 1980. <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig1.jpg">Para ver uma imagem maior clique aqui.</a> <img src="/files/ftp_upload/3084/3084fig2.jpg" alt="Figura 2" /> Figura 2. Desenvolvimento do total-IgY na gema de ovo de galinhas imunizadas na dependência de idade. Dado é a média semanal do total IgY / ovo e SD (n = 4 galinhas poedeiras). A partir de [Pauly et al. 2009]). <img src="/files/ftp_upload/3084/3084fig3.jpg" alt="Figura 3" /> Figura 3. Layimonitoramento capacidade ng de quatro galinhas imunizadas com diferentes antígenos. As setas indicam data de vacinação (de [Pauly et al. 2009]). <a href="http://www.jove.com/files/ftp_upload/3084/3084largefig3.jpg">Para ver uma imagem maior clique aqui.</a> <img src="/files/ftp_upload/3084/3084fig4.jpg" alt="Figura 4" /> Figura 4. Poliacrilamida Gelelectrophoresis de preparações individuais IgY de ovos diferentes sob condições redutoras Pista 1 - marcador de peso molecular, Lane 2 - IgY-padrão, Lane 05/03 - IgY amostras diferentes preparado como descrito no protocolo. Estes são os últimos preparativos de acordo com o passo 10 do protocolo. HC - cadeias pesadas, LC - chaines luz,? - Impurezas menores correspondente ao peso molecular em torno de 35 kDa (provavelmente o fragmento C-terminal de vitelogenina precursor II, [Klimentzou et al, 2006.]). <table border="1"><tbody><tr><td>&nbsp;</td><td> Chicken 21 (A Antigen) </td><td> Chicken 22 (um antígeno) </td><td> Chicken 19 (B Antigen) </td><td> Chicken 23 (C Antigen) </td></tr><tr><td> Número de ovos (IgY total) 1, 2 anos-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> Número de ovos (IgY total), primeiro ano </td><td> 345 (20 g) </td><td> 304 (16 g) </td><td> 326 (18 g) </td><td> 308 (17 g) </td></tr><tr><td> Número de ovos (IgY total), segundo ano </td><td> 280 (18 g) </td><td> 322 (21 g) </td><td> 219 (15 g) </td><td> 300 (20 g) </td></tr><tr><td> % Do valor máximo. número de ovos possível (primeiro ano) 2 </td><td> 106 </td><td> 93 </td><td> 100 </td><td> 95 </td></tr><tr><td> % Do valor máximo. número de ovos possível (segundo ano) </td><td> 86 </td><td> 99 </td><td> 67 </td><td> 92 </td></tr><tr><td> número de ovos processados </td><td> 565 </td><td> 620 </td><td> 283 </td><td> 401 </td></tr></tbody></table> 1 A quantidade de IgY total é calculado pela multiplicação do número de ovos por um valor médio de 60 mg de proteína por ovo de acordo com os dados da Figura 2. O 2 max. número de ovos por ano é possível 325 de acordo com uma informação do criador. Tabela 1. Estatísticas Dois anos de capacidade de postura de ovos de galinhas imunizadas com diferentes antígenos. Deitado capacidade em por cento do máximo de quatro galinhas após a imunização com antígenos diferentes, bem como o resultado IgY durante dois anos (ver também a fig. 3), [Pauly et al. 2009]). <table border="1"><tbody><tr><td> Nr ovo. </td><td> Nr galinha. Deitado Data </td><td> V1 [ml] yolk </td><td> V2 [ml] PBS </td><td> 1. PEG prec. [G] </td><td> Vol [ml] após </td><td> 2. PEG prec. [G] </td><td> Pastilha </td><td> 3. PEG prec. [G] </td><td> Pastilha </td><td> Vol [ml] </td><td> Proteínas totais mg / ml </td><td> Corrigida de proteína total 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. V3 e filtração </td><td> 8,5% [w / v] V3 </td><td> dissolvido em PBS ml V4 </td><td> 12% [w / v] V4 </td><td> Dissolvido em PBS ml V5 </td><td> Após diálise 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 / 23h10. </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> Tabela 2. Protocolo Exemplar de PEG precipitação

<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>

Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo pêlo Landesamt Gesundheit und Soziales Berlin (H 0069/03).

A escolha de um método adequado de purificação de IgY é influenciado pela escala de purificação (laboratório ou industrial), a eficácia de custos, tecnologia (equipamentos de laboratório), eo impacto sobre o meio ambiente (gestão de resíduos). Vários métodos de extração IgY foram revistos em detalhe pelo De Meulenaer &amp; Huyghebaert (2001, para revisão ver também Schade et al. 2005). Em geral, estes métodos podem ser divididos em três grupos principais: <ol><li> Métodos de precipitação: envolvendo amônio ou sulfato de sódio, polietilenoglicol (PEG), ácido caprílico e caragenean. </li><li> Métodos cromatográficos: cromatografia de afinidade, cromatografia de troca iônica, cromatografia de interação hidrofóbica, cromatografia de interação thiophylic e gel-filtração cromatografia. </li><li> Ultrafiltração. </li></ol> A pureza de uma preparação IgY pode ser aumentada por uma combinação de métodos, por exemplo, PEG precipitação pode ser combinada com cromatografia de afinidade. Em alguns casos, dependendo da aplicação final, um extrato de água de IgY é suficiente para atingir bons resultados (Akita &amp; Nakai 1993). De acordo com nossa experiência, a IgY-amostra obtemos por PEG-precipitação funcionou muito bem em um monte de diferentes ensaios imunológicos. Ao padronizar o método PEG-precipitação um assistente técnico é capaz de processar cerca de 84 ovos por semana, o que corresponde a uma quantidade de IgY total entre 4 e 7 g por semana. Um cálculo simples: uma galinha poedeira é imunizados com um antígeno. Depois de um impulso imunizações poucos a galinha produz anticorpos específicos (Ab) com um título de 1:50.000 durante um período de 30 dias. 30 ovos x 2 ml IgY extract (ver protocolo e fig. 3) corresponde a 60 ml IgY total, que por sua vez corresponde a uma diluição Ab-de 3.000 l (a capacidade de um balde é 10 l), isso significa que com 3.000 l de solução de Ab 300 baldes podem ser preenchidos. Assim, em vista de tal enorme quantidade Ab-it não é problema para armazenar um Ab-pool de qualidade constante e especificidade. Esta técnica reduz a variabilidade de carga / lote de outra forma observado em poliespecífico Ab. Apesar do aspecto de quantidade, IgY Ab têm vantagens adicionais em comparação com IgG de mamíferos: Não ativação do sistema complemento em mamíferos, sem reatividade cruzada com HAMA (anticorpo anti rato humano), fatores reumatóide ou antígenos no sangue humano em grupo (falta de heteroagglutinins). Uma vantagem notável de IgY-Ab é a distância filogenética so-called. Distância filogenética é a razão para as diferenças freqüentemente descritos entre as especificidades Ab de mamíferos e aves, mesmo quando idêntica imunizados. Além das diferenças entre os mamíferos e do sistema imunológico das aves si, diferenças de desenvolvimento filogenético nestas duas classes de animais contribui para as diferentes especificidades Ab. Vários autores têm relatado que as galinhas produzem frequentemente Abs contra filogeneticamente altamente conservada de proteínas de mamíferos ou peptídeos com mais eficiência do que os coelhos (para revisão ver Schade et a. 2005). Como conseqüência, um antígeno conservada pode permanecer &quot;mascarados&quot; para o sistema imunológico de coelho, e, portanto, causar apenas um fraco ou uma resposta &quot;silenciosa&quot;. Além disso, se galinhas e coelhos são imunizados com o mesmo antígeno de mamíferos, muitas vezes os frangos respondem com uma especificidade Ab-que raramente pode ser alcançado em coelhos. Também o aspecto de bem-estar animal é importante, pois o Ab não-invasiva são extraídos de gema de ovo. Em combinação com Gene Gun-imunização (plasmídeo), o IgY-produção é totalmente não-invasiva (Witkowski et al. 2009). Em conclusão, a produção de Ab em galinhas e as IgY-extração por meio de PEG-precipitação é muito custo-efetiva e resulta em Ab altamente específicas com títulos estáveis ​​até 1:1.000.000. Devido à distância filogenética entre Aves e Mamíferos, frango são capazes de produzir Ab específica contra antígenos altamente conservada em mamíferos, em contraste com os coelhos, por exemplo. A extraordinária quantidade de Ab IgY obtidos por tecnologia abre as portas também para a utilização de IgY-Ab na medicina humana e veterinária para fins terapêuticos / profilático.

<ol><li> Dialyse Visking Bag, Tipo 27/32, corte -14 kD, Carl Roth GmbH, Karlsruhe, Alemanha </li><li> Falcon tubos Blue Max, 50 ml de polipropileno tubo cônico, Becton Dickinson para laboratórios, Franklin Lakes NY, EUA </li><li> Dobrado Filtro MN 615 ¼, ∅ 150 mm, Carl Roth GmbH, Karlsruhe, Alemanha Heraeus </li><li> Agitador magnético, Variomag Mono, H + P Labortechnik AG, Oberschleißheim, Alemanha </li><li> Micro-dialyse-Capsule 5,0 ml, Carl Roth GmbH, Karlsruhe, Alemanha </li><li> Multifuge 3SR +, Thermo Scientific, Langenselbold, Alemanha </li><li> Fotômetro UV-160A, Shimadzu Deutschland GmbH, Duisburg, Alemanha </li><li> 6000 polietileno glicol, Rotipuran Ph. Eur., Carl Roth GmbH, Karlsruhe, Alemanha </li><li> Pré-misturado PBS-Buffer 10x, Roche Diagnostics GmbH, Mannheim, Alemanha </li><li> Rolo Mixer SRT2, Stuart, Staffordshire, UK </li><li> Vibrofix VF2, Janke &amp; Kunkel, IKA-Labortechnik, Staufen i.Br., Alemanha </li><li> Gema colher, Fackelmann (efeitos de uso doméstico), Alemanha </li></ol>

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

Galinhas podem ser vacinadas, por meio de im vacinação (Musculus peitoral, esquerda e direita, o volume de injeção de 0,5-1,0 ml) ou por meio de Gene Gun-imunização plasmídeos. Dependente da imunogenicidade do antígeno, alto-anticorpo títulos (até 1:100.000 - 1:1.000.000) pode ser obtida após apenas uma ou 3-4 imunizações impulso. Normalmente, uma galinha põe ovos continuamente por cerca de 72 semanas, depois, a diminui a capacidade de postura. Este protocolo descreve a extração de IgY totais de gema de ovo, por meio de um processo de precipitação (PEG. Polson et al. 1980). O método envolve duas etapas importantes. O primeiro é a remoção de lipídios ea segunda é a precipitação de IgY totais do sobrenadante de um passo. Após diálise contra um buffer (normalmente PBS) o extrato de IgY-podem ser armazenadas a -20 ° C por mais de um ano. A pureza do extrato é de cerca de 80%, o total por IgY de ovos varia de 40-80 mg, dependendo da idade da galinha poedeira. Os aumentos IgY totais de conteúdo com a idade da galinha de cerca de 40 mg / ovo até 80 mg / ovo (PEG sobre precipitação). A capacidade de postura de uma galinha por ano é de cerca de 325 ovos. Isso significa que uma colheita total potencial de 20 g IgY total / ano, com base em um conteúdo IgY média de 60 mg total de ovos IgY / (ver Tabela 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 descreve em particular o de extração de IgY totais de gema de ovo, por meio de precipitação polietileno glicol e dá informações gerais sobre a tecnologia IgY.

Tecnologia IgY: Extração de anticorpos de gema de ovo de galinha por polietileno glicol (PEG) Precipitação

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

64.1K visualizações.  Robert Koch-Institute. Este protocolo descreve em particular o de extração de IgY totais de gema de ovo, por meio de precipitação polietileno glicol e dá informações gerais sobre a tecnologia IgY.

Vídeo: 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.

Reconhecimento da transglutaminase epidérmica por 2 Anticorpos IgA transglutaminase tecidular e em um caso raro de Rhesus Dermatite

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

Imunologia e Infecção

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.

Um Protocolo de Chelex simples para extração de DNA 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.

Detecção de aspergilose pulmonar invasiva em pacientes com doença hematológica maligna usando fluxo lateral Tecnologia

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.

Purificação e visualização de lipopolissacarídeo de bactérias Gram-negativas por extracção aquosa de fenol-Hot

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.

Propagação do vírus Influenza em ovos de galinha embrionados

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.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

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.

Geração de anticorpos monoclonais murinos pelo hibridoma 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.

Um método para avaliar funções efetoras mediada por Fc induzidas por anticorpos específicos de hemaglutinina de 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.

Geração dos anticorpos monoclonais humanos discriminativa de rara B antígeno-específicas células circulantes no 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.

Extração de hemócitos de larvas de Drosophila melanogaster para infecção microbiana e análise

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