The authors thank Miss Ching-Chun Lin and Miss Diana Lin of the Core Facility of the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica for offering their expertise on peptide synthesis and DNA sequencing, respectively. This study was supported by Academia Sinica.

1. Preparação de Anticorpos Monoclonais <ol><li> Cultura dos monoclonais de rato células de hibridoma RG-M56 2 em meio livre de soro em frascos de 175T a 37 ºC com 5% de suplemento de CO 2. Recolhe-se o sobrenadante quando a cor do meio torna-se amarelo após cinco dias de incubação. NOTA: As células de hibridoma foram cultivadas em meio isento de soro a fim de evitar a contaminação de anticorpos a partir de soro fetal de bovino. </li><li> Centrifuga-se o sobrenadante a 4500 xg durante 30 min a 4 ºC e descartar o sedimento de restos de células. </li><li> Adicionar 2 ml de agarose a proteína G (fornecido como uma pasta a 50%) a uma coluna de 5 mL e equilibrar-se com 10 volumes de resina (10 ml) de PBS gelado. </li><li> Carga de 200 ml de sobrenadante de anticorpo (passo 1.2) para a coluna e descartar o repasse. </li><li> Adicionam-se 10 mL de PBS arrefecido em gelo para a coluna de lavá-lo. Repita duas vezes. </li><li> Adicionar 10 ml de glicina 50 mM, pH 2,7 à coluna para eluir o anticorpo da proteína G-associado. Colcionar 900 fracções ul num tubo de microcentrífuga contendo 100 pL de 10x tampão de neutralização (Tris 1 M, NaCl 1,5 M, e EDTA 1 mM, pH 8,0). </li><li> Armazenar o anticorpo purificado em glicerol a 50% com 0,03% de NaN3 a -20 ºC. </li></ol> 2. Construção e Expressão da série truncadas Proteínas Recombinantes <ol><li> Prepara-se uma mistura reaccional de PCR: 5? L de 10x tampão Pfu, 0,2 mM de cada dNTP, 0,2 uM de iniciadores para a frente 3, 0,2 fiM de primer reverso 3, MgSO 2 mM de 4, 1 ng de pET20b 1A59 ADN-3 plasmídeo, e 2,5 L ( unidade) de polimerase de ADN Pfu; adicionar ddH2O para um volume final de 50 uL. <ol><li> amostras de execução em um termociclador automático, utilizando os seguintes parâmetros: Ciclo 1 (94 ºC por 5 min); ciclos 2-36 (94 ºC durante 30 s, 63 ° C durante 30 s, e 72 ºC durante 60 s); e ciclo de 37 (72 ºC durante 7 min). </li></ol></li><li> Extrai-se a reacção em cadeia da polimerase (PCR) produtos usando um kit de purificação de PCR para facilitar a 5 após a digestão com enzimas de restrição. </li><li> Digerir os fragmentos de ADN amplificados por PCR com Ndel e Xhol as enzimas de restrição e ligar cada um destes fragmentos de ADN Ndel e Xhol clivado-enzima pET-20b (+) do vetor. Transformar as construções em células de Escherichia coli-DH-5a competente 6. <ol><li> Preparar a mistura de digestão em tampão de digestão (20 mM Tris-acetato, Mg 10 mM (CH3COO) 2, KCH 50 mM 3 COO, e DTT 1 mM, pH 7,9) com 1 ug de ADN amplificado por PCR ou pET-20b (+) DNA do vector, e 2 U de Ndel e Xhol as enzimas de restrição, num volume final de 20 uL. </li><li> Misture a mistura de digestão suave e rapidamente girar para baixo. Incubar a 37 ° C durante 2 h num banho seco de corte para assegurar a completa da restrição sentares. </li><li> Extrair os fragmentos de ADN digerido com enzimas de restrição, utilizando um kit de purificação de PCR 5 para facilitar a seguinte construção de plasmídeo. </li><li> Preparar a mistura de ligação em tampão de ligação (Tris 66 mM, MgCl2 5 mM, ATP 1 mM, e DTT 5 mM, pH 7,5) com 100 ng de ADN pré-digeridas, amplificado por PCR, 10 ng de pré-digerido pET-20b (+) ADN do vector, e 5 L de ligase de ADN de T4 num volume final de 10 uL. </li><li> Misture a mistura de ligação com cuidado e rapidamente girar para baixo. Incubar a 16 ° C durante 18 h num banho de água. </li><li> Coloque 10 uL das amostras de ligação em 100 ul de células DH-5α-competentes e misturar suavemente antes da colocação do tubo de microcentrífuga em gelo durante 30 min. Coloque o tubo de microcentrífuga em um banho seco a 42 ºC durante 90 s para induzir choque de calor 7. Imediatamente transferir o tubo em gelo durante 2 min. </li><li> Adicionar 900 uL de meio de Luria-Bertani (LB) (1% de Bacto-triptona, 0,5% de Bacto levedura extracT, e 0,5% de NaCl, pH 7,0) ao tubo. Incubar a 37 ° C com agitação 150 rpm durante 45 min. Agregar as células por centrifugação a 4000 x g durante 10 min e desprezar o sobrenadante. </li><li> Ressuspender o sedimento com 50 ul de caldo LB e espalhar cada transformação em placas pré-aquecido LB contendo 100 ug / ml de ampicilina. Incubar as placas a 37 ºC durante 16 h. </li><li> Pegar uma única colónia usando uma ponta de 200 mL e colocá-lo em caldo 3 ml de LB contendo 100 ug / ml de ampicilina em um tubo de 15 frouxamente tapado mL. Incubar a 37 ° C com agitação 150 rpm durante 12 h. </li><li> Extrai-se a ADN de plasmídeo a partir de 8 cada cultura e sequenciar-lo utilizando um promotor de T7 e iniciadores de terminador T7 para confirmar a sequência 9. </li></ol></li><li> Após confirmação da sequência, transformar 10 ng de DNA de pET-20b estes (+) plasmídeos com diferentes comprimentos de gene da proteína de revestimento YGNNV na estirpe BL-21 (DE3) de E. coli por nósing o método de choque de calor 7. Siga os passos 2.3.6-2.3.8 para realizar a transformação. </li><li> Transferir uma única colónia de cada um de E. coli transformada BL-21 de células em caldo de 3 ml de LB contendo 100 ug / ml de ampicilina num tubo de 15 ml tapado frouxamente. Incubar a cultura a 37 ° C com 150 rpm tremendo. </li><li> Arrefece-se a cultura a 25 ° C quando a OD600 da cultura é de cerca de 0,6 e adicionar IPTG para uma concentração final de 0,4 mM para induzir a expressão da proteína recombinante. Incubar a cultura para um extra de 4 h a 25 ºC com 200 rpm tremendo. </li><li> Transferir 1 ml da cultura para um tubo de microcentrífuga. Agregar as células por centrifugação a 12000 xg durante 1 min e desprezar o sobrenadante. </li><li> Ressuspender o sedimento celular em 100 uL de tampão de desnaturação (ureia 8 M, fosfato de sódio 20 mM, e NaCl 0,5 M, pH 7,4) por pipetagem para cima e para baixo com uma micropipeta. Misturar em vórtice vigoroso. NOTA: O sol da amostradoras deve agora ser semi-transparente, um pouco pegajoso, e pronto para o ensaio de hibridação dot-blot. </li></ol> 3. Projeto e Síntese de péptidos sobrepostos <ol><li> Concepção e sintetizar 3 série peptídeos 20-meros que cada coincidem com o seu sucessor por 10 resíduos de aa da região de 195-338 aa da proteína de revestimento YGNNV para diminuir a região do epitopo de RG-M56 mAb por transferência de pontos. </li><li> Concepção e sintetizar peptídeos 3 três 8-Mer (195 VNVSVLCR 202, 197 VSVLCRWS 204, e 199 VLCRWSVR 206) com uma sobreposição de 6 resíduos aa para o próximo peptídeo sintético para diminuir a região do epitopo de 195-206 aa por transferência de pontos. </li><li> Concepção e sintetizar 3 7-mer (196 NVSVLCR 202 e 195 VNVSVLC 201), 6-mer (195 VNVSVL 200, 196 NVSVLC 201, e 197 VSVLCR 202), e 5-meros péptidos (195 VNVSV 199, 196 NVSVL 200, 197 VSVLC 201, e 198 SVLCR 202) com uma sobreposição de 6, 5, e 4 resíduos de aa, respectivamente, Onto seus péptidos vizinhos para minimizar o região de epítopo por dot blotting. </li></ol> 4. Dot-blot hibridação <ol><li> Dissolve-se cada péptido sintetizado em sulfóxido de dimetilo (DMSO) a uma concentração final de 10 mg / mL. NOTA: Para superar a solubilidade variada de péptidos sintéticos, todos os péptidos sintéticos devem ser dissolvidos em DMSO. DMSO é um bom solvente para dissolver completamente péptidos hidrofóbicos ou hidrofílicos. </li><li> Embeber a membrana de fluoreto de polivinilideno (PVDF) com metanol durante 2 min. NOTA: membrana PVDF é até resistente a DMSO 100%; outros podem não ser assim. </li><li> Equilibrar a membrana de PVDF com tampão Towbin modificado (Tris 25 mM, glicina 192 mM e SDS a 0,1%, pH 8,3) para2 minutos. NOTA: 10-20% (v / v) de metanol pode ser adicionado ao tampão Towbin modificados para melhorar os resultados de transferência. </li><li> Lavar um pedaço de papel de cromatografia com o tampão Towbin modificado. Colocar a membrana de PVDF sobre o papel de cromatografia. Espere até que o tampão Towbin modificado tenha desaparecido da superfície da membrana de PVDF antes de continuar para a próxima etapa. </li><li> Adicionar 2 mL de cada amostra de péptido para a membrana com uma ponta de 10 uL. Ar seco a membrana de PVDF sobre o papel de cromatografia durante 10 min. Adicionar a cada amostra de péptido lenta e gradualmente para a membrana para evitar o excesso de difusão. </li><li> Bloquear a membrana em tampão TBST (0,05% (v / v) de Tween-20, Tris 20 mM, e NaCl 150 mM, pH 7,4) com leite magro a 5% durante 30 min à temperatura ambiente com agitação suave. </li><li> Adicionar RG-M56 mAb a uma diluição final de 1: 1000 em tampão TBST com 5% de leite desnatado à membrana. Incubar a membrana a 37 ° C durante 1 h com agitação suave. </li><li> Remover o anticorpo assimlução. Lava-se a membrana em tampão TBST durante 5 min com agitação suave. Repita duas vezes. </li><li> Adicionar o anticorpo secundário (anticorpo de cabra anti-IgG de ratinho, Fe, conjugado com fosfatase alcalina) numa diluição final de 1: 5000 em tampão TBST com 5% de leite desnatado à membrana. Incubar a membrana a 37 ° C durante 1 h com agitação suave. </li><li> Descartar a solução do anticorpo. Lava-se a membrana em tampão TBST durante 5 min com agitação suave. Repita o passo de lavagem duas vezes. </li><li> Desenvolver a membrana com uma solução de substrato BCIP / NBT temperatura ambiente durante 15 min no escuro. Parar o desenvolvimento por lavagem da membrana com ddH2O quando o sinal aparece. </li><li> Ar seco da membrana e capturar a imagem dot-blot utilizando um sistema de imagem. </li><li> Medir a intensidade de cada dot blot usando uma imagem análise de software 3. </li></ol> 5. varrimento de alanina e Substituição <ol><li> Projetar e sintetizar 3 alanina e methipeptídeos de substituição onine. Substituir cada resíduo aa com alanina para o péptido 8-mer 195 VNVSVLCR 202 e sintetizar peptídeos 195 ANVSVLCR 202, 195 VAVSVLCR 202, 195 VNASVLCR 202, 195 VNVAVLCR 202, 195 VNVSALCR 202, 195 VNVSVACR 202, 195 VNVSVLAR 202 e 195 VNVSVLCA 202 . Substitua resíduo aa leucina 200 a metionina para criar o péptido epitópico SJNNV genótipo 195 VNVSVMCR 202. </li><li> Siga Seção 4 para realizar pesquisa de alanina e mutagénese de substituição dot blot. </li></ol>

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The authors have no conflicts of interest related to this report.

Este protocolo oferece uma técnica rápida e simples para identificar um epitopo linear reconhecido-mAb. Tomando em consideração o custo da síntese de péptidos e da eficiência da produção de péptidos que sintetizam, da região antigénica da proteína de revestimento de vírus foi reduzido por expressão de proteínas recombinantes em série truncados antes da análise de varrimento de péptidos. Como tal, o sistema de expressão pET de E. coli fiável e eficiente foi usado para produzir estas proteín...

No sistema imunitário, a recombinação de segmentos V, D, J e permite a anticorpos para criar grandes variações de regiões determinantes de complementaridade (CDR) para se ligar a vários antigénios para proteger o hospedeiro contra a infecção patogénica. A defesa de neutralização de anticorpos contra antigénios depende da complementaridade espacial entre as CDR dos anticorpos e os epítopos de antigénios. Portanto, o conhecimento desta interacção irá ajudar molecular concepção de uma vacina profilática e desenvolvimento de fármacos péptido terapêutico. No entanto, esta interacção de neutralização pode ser influenciada tanto por vários domínios antigénicos a partir de um único antigénio e por vários CDRs de anticorpos, os quais, por conseguinte, tornam o processo de determinação epitopo mais complexo. Felizmente, o desenvolvimento da tecnologia do hibridoma, o qual funde a células produtoras de anticorpos com células de mieloma individuais, permite um lote constante dividindo de células para segrete um anticorpo específico, conhecido como um anticorpo monoclonal (mAb) 1. As células de hibridoma produzem estes puros, mAb de elevada afinidade para se ligar a um único domínio antigénico de um antigénio específico. Com a relação do anticorpo-antigénio estabelecida, várias abordagens, incluindo varrimento de péptidos, pode ser utilizado para determinar o epítopo de um antigénio usando o mAb correspondente. Desenvolvimentos recentes na tecnologia peptídeo sintético fizeram a técnica de exploração peptídeo mais acessível e mais conveniente para executar. Resumidamente, uma série de péptidos sintéticos que se sobrepõem são produzidos de acordo com uma sequência do antigénio alvo e estão associados a uma membrana em suporte sido para a hibridação de mAb. digitalização péptido não só oferece uma maneira simples para mapear a região de ligação do anticorpo, mas também facilita o aminoácido (AA) por meio de mutagénese de varrimento resíduo ou substituição para avaliar a interacção de ligação entre cada resíduo AA do péptido epítopo e as CDR do anticorpo. <p class = &quot;jove_content&quot;&gt; Aqui, o presente estudo descreve um protocolo para a identificação eficiente do epitopo linear da proteína de revestimento garoupa amarelo vírus da necrose nervoso (YGNNV) usando um mAb neutralizante 2, 3, 4. O protocolo inclui mAb preparação, a construção e expressão de proteínas recombinantes em série truncadas, de criação de péptidos sintéticos sobrepostos, hibridação dot-blot, o varrimento de alanina, e mutagénese de substituição. Considerando o elevado custo da síntese de péptidos, o passo de truncagem em série das proteínas recombinantes de uma proteína alvo desejado foi modificado, e a região antigénica foi reduzida para cerca de 100 a 200 resíduos aa antes da análise dot-blot matriz peptídica sintética foi realizada.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hybrid-SFM medium</td>
			<td>Gibco</td>
			<td>12045-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos&#39;s Phophate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Pfu DNA Polymerase&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>EP0502&#160;</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>XhoI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td>Including buffers</td>
		</tr>
		<tr>
			<td>pET-20b(+) vector</td>
			<td>Novagen, Merck Millipore</td>
			<td>69739</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli DH-5&#945; competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH617</td>
			<td></td>
		</tr>
		<tr>
			<td>E.coli BL-21(DE3) competent cell</td>
			<td>RBC Bioscience</td>
			<td>RH217</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Amresco</td>
			<td>0339-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth&#160;</td>
			<td>Invitrongen</td>
			<td>12780-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropylthio-&#946;-D-thiogalactoside (IPTG)</td>
			<td>MDBio, Inc.</td>
			<td>101-367-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck Millipore</td>
			<td>106009</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T.Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Amresco</td>
			<td>0167-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Affymetrix, USB</td>
			<td>75825</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Amresco</td>
			<td>0241-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Amresco</td>
			<td>0105-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Amresco</td>
			<td>0854-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>NaN3</td>
			<td>Sigma</td>
			<td>S2002-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP/NBT</td>
			<td>PerkinElmer</td>
			<td>NEL937001PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG, Fc fragment antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-055-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P (Polyvinylidene fluoride, PVDF)</td>
			<td>Merck Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein G Agarose Fast Flow</td>
			<td>Merck Millipore</td>
			<td>16-266</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP BioSpectrum 600 Image System</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>VisionWorks LS Analysis Software Ver 6.8</td>
			<td>UVP</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>MyCycler thermal cycler</td>
			<td>BioRad</td>
			<td>1709713</td>
			<td></td>
		</tr>
	</tbody>
</table>

peptide scanning-assisted identification of a monoclonal antibody-recognized linear b-cell epitope

A identificação de um epítopo antigénico pelo sistema imunológico permite a compreensão do mecanismo de protecção de anticorpos que podem facilitar o desenvolvimento de vacinas e de drogas peptídicas neutralizantes. Péptido de varrimento é um método simples e eficiente, que diretamente mapeia o epitopo linear reconhecido por um anticorpo monoclonal (mAb). Aqui, os autores apresentam uma metodologia de determinação epitopo envolvendo proteínas em série truncadas recombinantes, design peptídeo sintético, e hibridação dot-blot para o reconhecimento antigênico da proteína de revestimento do vírus da necrose nervoso usando um mAb neutralizante. Esta técnica baseia-se na hibridação dot-blot de péptidos sintéticos e os mAbs em um (PVDF) de membrana de fluoreto de polivinilideno. A região mínima antigénico de uma proteína de revestimento virai reconhecido pelo mAb M56-RG pode ser reduzida por passo-a-passo aparado mapeamento do péptido para um epitopo peptidico 6-mer. Além disso, a mutagénese de varrimento de alanina e o resíduo subtuição pode ser realizado para caracterizar a importância de ligação de cada resíduo de aminoácidos que constituem o epítopo. Os resíduos que flanqueiam o local de epitopo foram encontrados para jogar um papel crítico na regulação da conformação peptídica. O péptido epitopo identificado podem ser usadas para formar cristais de complexos de péptido-anticorpo epitopo para um estudo de difracção de raios-X e a competição funcional, ou para terapêutica.

O objetivo deste experimento foi identificar um epitopo através Dot blot usando mAb. Para limitar rapidamente e eficientemente para baixo da região antigénica reconhecido por mAb, a todo o comprimento e proteínas de revestimento recombinante YGNNV truncados em série com uma marcação de fusão 6xHis no terminal C foram expressos a partir de um sistema de expressão de E. coli de PET 10 (Figura 1A). As proteínas recombinantes result...

Watch this Scientific Journal Video about Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope at JoVE.com

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Identificação de um epítopo de célula B linear Monoclonal Antibody-péptido reconhecido Digitalização-assistida

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

peptide-scanning-assisted-identification-monoclonal-antibody

Research

JoVE Journal

Immunology and Infection

9.3K visualizações.  Academia Sinica. Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

Vídeo: Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Péptido: Enriquecimento tetrâmero com base MHC das células T específicas para epitopos

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Imunologia e Infecção

Peptide-based Identification of Functional Motifs and their Binding Partners

Specific short peptides derived from motifs found in full-length proteins, in our case HIV-1 Nef, not only retain their biological function, but can also competitively inhibit the function of the full-length protein. A set of 20 Nef scanning peptides, 20 amino acids in length with each overlapping 10 amino acids of its neighbor, were used to identify motifs in Nef responsible for its induction of apoptosis. Peptides containing these apoptotic motifs induced apoptosis at levels comparable to the full-length Nef protein. A second peptide, derived from the Secretion Modification Region (SMR) of Nef, retained the ability to interact with cellular proteins involved in Nef's secretion in exosomes (exNef). This SMRwt peptide was used as the "bait" protein in co-immunoprecipitation experiments to isolate cellular proteins that bind specifically to Nef's SMR motif. Protein transfection and antibody inhibition was used to physically disrupt the interaction between Nef and mortalin, one of the isolated SMR-binding proteins, and the effect was measured with a fluorescent-based exNef secretion assay. The SMRwt peptide's ability to outcompete full-length Nef for cellular proteins that bind the SMR motif, make it the first inhibitor of exNef secretion. Thus, by employing the techniques described here, which utilize the unique properties of specific short peptides derived from motifs found in full-length proteins, one may accelerate the identification of functional motifs in proteins and the development of peptide-based inhibitors of pathogenic functions.

Techniques to dissect the mechanisms underlying the secretion of HIV-1 Nef in exosomes are described. Specific short peptides derived from Nef and protein transfection were exploited to determine the structure, function, and binding partners of Nef&#x2019;s Secretion Modification Region. These procedures have general relevance in many mechanistic studies.

Identificação baseada em peptídeo de motivos funcionais e seus parceiros de ligação

Specific short  peptides derived from motifs found in full-length proteins, in our case HIV-1  Nef, not only retain their biological function, but can ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Isolamento de precursor de células B subconjuntos de Sangue do Cordão Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Geração de variantes de fuga de neutralizar os anticorpos monoclonais de vírus da gripe

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

Purificação de DNA Viral para a identificação de proteínas Viral e celulares associadas

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules play important roles in surveying cells for structural and functional integrity, inducing immune regulation, and limiting immune responses to viral infections. Additionally, functional augmentation of Qa-1-restricted CD8+ T cells through epitope immunization has shown therapeutic effects in several autoimmune disease animal models, e.g. experimental allergic encephalomyelitis, collagen-induced arthritis, and non-obese diabetes. Therefore, there is an urgent need for a method that can efficiently and quickly identify functional Qa-1 epitopes in a protein. Here, we describe a protocol that utilizes Qa-1-restricted CD8+ T cell lines specific for an overlapping peptide (OLP) library for determining Qa-1 epitopes in a protein. This OLP library contains 15-mer overlapping peptides that cover the whole length of a protein, and adjacent peptides overlap by 11 amino acids. Using this protocol, we recently identified a 9-mer Qa-1 epitope in myelin oligodendrocyte glycoprotein (MOG). This newly mapped MOG Qa-1 epitope was shown to induce epitope-specific, Qa-1-restricted CD8+ T cells that enhanced myelin-specific immune regulation. Therefore, this protocol is useful for future investigation of novel targets and functions of Qa-1-restricted CD8+ T cells.

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b molecules. Immunization with Qa-1-binding epitopes has been shown to augment tissue-specific immune regulation and ameliorate several autoimmune diseases. Herein we describe an overlapping peptide library strategy for the identification of Qa-1 epitopes in a protein.

Biblioteca de peptídeo sobreposição para mapear os resumos de Qa-1 em uma proteína

Qa-1 (HLA-E in human) belongs to a group of non-classical major histocompatibility complex 1b (MHC-Ib) molecules. Recent data suggest that Qa-1 molecules ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

In this protocol, we describe a technique to discover Zika virus specific diagnostic peptides using a high-density peptide microarray. This protocol can readly be adapted for other emerging infectious diseases.

Zika vírus específico diagnóstico Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

Geração de anticorpos monoclonais contra produtos naturais

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...