Name: a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor
Uploaded: 2011-06-27T13:00:00
Description: Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

Estamos muito gratos a K. Nakagaki, H. Dr. Ishii, Dr. K. Suzuki, Yamagata A., K. Oofusa por suas valiosas contribuições.

1. Produção e purificação de sGMRα <ol><li> Amplificar cDNA de sGMRα da biblioteca de cDNA placenta humana por reação em cadeia da polimerase (PCR). </li><li> Adicionar 50 base de 5&#39;-UTR seqüência de final poliedrina baculovírus e 27 RGS base de codificação Sua tag-de-final 3 &#39;do fragmento amplificado pela PCR por outro PCR. </li><li> Inserir o produto amplificado de PCR em um pMSG1.1MG plasmídeo. </li><li> Injetar o psGMR/M1.1MG construir com o vetor helper pHA3PIG em ovos de bicho-da-tensão pn...

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O ensaio livre de células estimou a capacidade de neutralização de GM-CSF auto-anticorpos com excelente reprodutibilidade e rapidez. A inibição de ligação por auto-anticorpos GM-CSF ou frações de soro do paciente IgG foi avaliada por este ensaio. Os dados mostraram uma correlação entre a inibição de ligação do ensaio livre de células ea inibição do crescimento de um bioensaio utilizando células TF-1, respectivamente. O bioensaio tem sido amplamente utilizada, mas abrigava dificuldades na comparação ...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

FUNDOS: Anteriormente, demonstramos que a capacidade de neutralizar, mas não a concentração de GM-CSF auto-anticorpos foi correlacionada com a severidade da doença em pacientes com proteinose alveolar pulmonar auto-imunes (PAP) 1-3. Como revogação da GM-CSF bioatividade no pulmão é a causa mais provável para PAP auto-imunes 4,5, é promissora para medir a capacidade de neutralização de GM-CSF auto-anticorpos para avaliar a gravidade da doença em cada paciente com PAP. Até agora, a capacidade de neutralização de GM-CSF auto-anticorpos foi avaliada através da avaliação da inibição do crescimento de células de medula óssea humana ou TF-1 células estimuladas com GM-CSF 6-8. No sistema de bioensaio, no entanto, muitas vezes é problemático para obter dados fiáveis, bem como comparar os dados de diferentes laboratórios, devido a dificuldades técnicas na manutenção das células em uma condição constante. OBJETIVO: Para imitar GM-CSF ligação ao GM-CSF receptor na superfície celular utilizando células livres receptor-ensaio de ligação. MÉTODOS: Transgênicos bicho-da-tecnologia foi aplicada para a obtenção de uma grande quantidade de recombinante solúvel GM-CSF receptor alfa (sGMRα) com alta pureza 9-13. O sGMRα recombinante foi contido nas camadas sericin hidrofílica de fios de seda sem ser fundida ao proteínas da seda, e assim, podemos facilmente extrair os casulos na pureza bom com soluções aquosas neutras 14,15. Felizmente, as estruturas de oligossacarídeos, que são críticos para a ligação com a GM-CSF, são mais semelhantes às estruturas de sGMRα humana do que aquelas produzidas por outros insetos ou leveduras. RESULTADOS: O sistema de ensaio livre de células usando sGMRα rendeu os dados com alta plasticidade e confiabilidade. GM-CSF ligação a sGMRα foi dose-dependente inibida por policlonal GM-CSF auto-anticorpos de forma semelhante ao bioensaio usando TF-1 células, indicando que o nosso novo livre de células do sistema de ensaio utilizando sGMRα é mais útil para a medição da atividade neutralizante de GM-CSF auto-anticorpos que o sistema de bioensaio utilizando TF-1 célula ou células humanas de medula óssea. CONCLUSÕES: Nós estabelecemos um ensaio livre de células quantificar a capacidade de neutralização da GM-CSF auto-anticorpos.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Nós projetamos um ensaio de receptor de células livres vinculativas, a fim de estimar a ligação de granulócitos e macrófagos fator estimulante de colônia (GM-CSF) para os receptores. Ele nos permite avaliar inibição competitiva do biotinilado GM-CSF ligação a solúvel GM-CSF receptor alfa por GM-CSF auto-anticorpos com excelente reprodutibilidade.

A célula do sistema de Ensaio grátis Estimar a capacidade de neutralização de GM-CSF recombinante usando anticorpo receptor GM-CSF Solúvel

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

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