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

Nous sommes très reconnaissants à K. Nakagaki, le Dr H. Ishii, le Dr K. Suzuki, A. Yamagata, K. Oofusa pour leur précieuse contribution.

1. Production et purification de sGMRα <ol><li> Amplifier l&#39;ADNc d&#39;sGMRα du placenta banque d&#39;ADNc humain en chaîne par polymérase (PCR). </li><li> Ajouter de base 50 5&#39;-UTR séquence de fin de polyhédrine de baculovirus et 27 RGS bases codant His-tag à l&#39;extrémité 3 &#39;de l&#39;amplicon PCR PCR par un autre. </li><li> Insérez le produit amplifié par PCR dans un plasmide pMSG1.1MG. </li><li> Injecter l&#39;psGMR/M1.1MG construire avec le vecteur d&#39;aide pHA3PIG dans des œufs de vers...

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Le test sans cellule estimé la capacité de neutralisation de GM-CSF auto-anticorps avec une excellente reproductibilité et de rapidité. L&#39;inhibition de la fixation par le GM-CSF autoanticorps ou le sérum du patient fractions d&#39;IgG a été évaluée par ce test. Les données ont montré une corrélation entre l&#39;inhibition de la fixation de l&#39;essai sans cellules et l&#39;inhibition de croissance d&#39;un test biologique utilisant cellules TF-1, respectivement. Le bio-essai a été largement utilisé, ...

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

Arrière-plans: Auparavant, nous avons démontré que la capacité de neutraliser, mais pas la concentration de GM-CSF d&#39;auto-anticorps a été corrélée avec la sévérité de la maladie chez les patients atteints protéinose alvéolaire auto-immune pulmonaire (PAP) 1-3. Comme l&#39;abrogation de GM-CSF bioactivité dans le poumon est la cause probable pour PAP auto-immunes 4,5, il est prometteur de mesurer la capacité de neutralisation des anticorps GM-CSF pour évaluer la sévérité de la maladie chez chaque patient avec PAP. Jusqu&#39;à présent, la capacité de neutralisation de GM-CSF auto-anticorps a été évaluée en évaluant l&#39;inhibition de la croissance de cellules humaines de la moelle osseuse ou cellules TF-1 stimulées par le GM-CSF 6-8. Dans le système de dosage biologique, cependant, il est souvent problématique pour obtenir des données fiables ainsi que de comparer les données de différents laboratoires, en raison des difficultés techniques dans le maintien des cellules dans un état constant. OBJECTIF: Pour imiter le GM-CSF se liant à des récepteurs du GM-CSF à la surface cellulaire utilisant des cellules sans liaison au récepteur-dosage. Méthodes: soie transgénique technologie a été appliquée pour obtenir une grande quantité d&#39;alpha recombinant soluble du récepteur GM-CSF (sGMRα) avec une grande pureté 9-13. Le sGMRα recombinante a été contenue dans les couches séricine hydrophile de fils de soie sans être fusionnée à la protéine de soie, et donc, nous pouvons facilement extraire à partir des cocons de bonne pureté avec des solutions aqueuses neutres 14,15. Heureusement, les structures d&#39;oligosaccharides, qui sont critiques pour la liaison avec le GM-CSF, sont plus semblables à des structures de sGMRα humaines que celles produites par d&#39;autres insectes ou les levures. RÉSULTATS: Le système de dosage sans cellule en utilisant sGMRα donné les données avec une grande plasticité et leur fiabilité. Contraignant pour sGMRα GM-CSF était dose-dépendante inhibée par le GM-CSF polyclonaux auto-anticorps dans une manière similaire à l&#39;essai biologique utilisant cellules TF-1, indiquant que notre nouveau système de dosage sans cellule en utilisant sGMRα est plus utile pour la mesure de l&#39;activité neutralisante de GM-CSF auto-anticorps que le système d&#39;essai biologique utilisant des cellules TF-1 ou de cellules osseuses humaines. CONCLUSIONS: Nous avons établi un test sans cellule de quantifier la capacité de neutralisation de GM-CSF autoanticorps.

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

Nous avons conçu un dosage des récepteurs cellulaires sans liant en vue d&#39;estimer la liaison de granulocyte-macrophage colony-stimulating factor (GM-CSF) pour les récepteurs. Il nous permet d&#39;évaluer l&#39;inhibition compétitive de biotinylé GM-CSF se liant à des récepteurs solubles de GM-CSF alpha par le GM-CSF d&#39;auto-anticorps avec une excellente reproductibilité.

Un système de dosage Free Cell Estimation de la capacité de neutralisation de GM-CSF recombinant soluble à l&#39;aide d&#39;anticorps GM-CSF récepteur

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

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