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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	<li>Arai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+neutralizing+capacity+of+GM-CSF+reflects+disease+severity+in+a+patient+with+pulmonary+alveolar+proteinosis+successfully+treated+with+inhaled+GM-CSF.">Serum neutralizing capacity of GM-CSF reflects disease severity in a patient with pulmonary alveolar proteinosis successfully treated with inhaled GM-CSF.</a> Respir Med. 98, 1227-1230 (2004).</li><li>Tazawa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+and+lung+immunity+in+pulmonary+alveolar+proteinosis.">Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis.</a> Am J Respir Crit Care Med. 171, 1142-1149 (2005).</li><li>Inoue, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+large+cohort+of+patients+with+autoimmune+pulmonary+alveolar+proteinosis+in+Japan.">Characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in Japan.</a> Am J Respir Crit Care Med. 177, 752-762 (2008).</li><li>Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+autoantibodies+specifically+eliminate+granulocyte-macrophage+colony-stimulating+factor+activity+in+the+lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis.">High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis.</a> Blood. 103, 1089-1098 (2004).</li><li>Sakagami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+GM-CSF+autoantibodies+and+reproduction+of+pulmonary+alveolar+proteinosis.">Human GM-CSF autoantibodies and reproduction of pulmonary alveolar proteinosis.</a> N Engl J Med. 361, 2679-2681 (2009).</li><li>Raines, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+molecular+cloning+of+a+soluble+human+granulocyte-macrophage+colony-stimulating+factor+receptor.">Identification and molecular cloning of a soluble human granulocyte-macrophage colony-stimulating factor receptor.</a> Proc Natl Acad Sci U S A. 88, 8203-8207 (1991).</li><li>Williams, W., VonFeldt, J., Rosenbaum, H., Ugen, K., Weiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+soluble+form+of+the+granulocyte-macrophage+colony-stimulating+factor+receptor+alpha+chain+from+a+myelomonocytic+cell+line.+Expression,+biologic+activity,+and+preliminary+analysis+of+transcript+distribution.">Molecular cloning of a soluble form of the granulocyte-macrophage colony-stimulating factor receptor alpha chain from a myelomonocytic cell line. Expression, biologic activity, and preliminary analysis of transcript distribution.</a> Arthritis Rheum. 37, 1468-1478 (1994).</li><li>Prevost, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granulocyte-macrophage+colony-stimulating+factor+(GM-CSF)+and+inflammatory+stimuli+up-regulate+secretion+of+the+soluble+GM-CSF+receptor+in+human+monocytes:+evidence+for+ectodomain+shedding+of+the+cell+surface+GM-CSF+receptor+alpha+subunit.">Granulocyte-macrophage colony-stimulating factor (GM-CSF) and inflammatory stimuli up-regulate secretion of the soluble GM-CSF receptor in human monocytes: evidence for ectodomain shedding of the cell surface GM-CSF receptor alpha subunit.</a> J Immunol. 169, 5679-5688 (2002).</li><li>Iizuka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+recombinant+mouse+monoclonal+antibody+in+transgenic+silkworm+cocoons.">Production of a recombinant mouse monoclonal antibody in transgenic silkworm cocoons.</a> FEBS J. 276, 5806-5820 (2009).</li><li>Iizuka, M., Tomita, M., Shimizu, K., Kikuchi, Y., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+enhancement+of+recombinant+protein+synthesis+in+transgenic+silkworms+by+a+5'-untranslated+region+of+polyhedrin+gene+of+Bombyx+mori+Nucleopolyhedrovirus.">Translational enhancement of recombinant protein synthesis in transgenic silkworms by a 5'-untranslated region of polyhedrin gene of Bombyx mori Nucleopolyhedrovirus.</a> J Biosci Bioeng. 105, 595-603 (2008).</li><li>Zou, W., Ueda, M., Yamanaka, H., Tanaka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+combinatorial+protein+library+displayed+on+yeast+cell+surface+using+DNA+random+priming+method.">Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method.</a> J Biosci Bioeng. 92, 393-396 (2001).</li><li>Tamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nat Biotechnol. 18, 81-84 (2000).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+silkworms+produce+recombinant+human+type+III+procollagen+in+cocoons.">Transgenic silkworms produce recombinant human type III procollagen in cocoons.</a> Nat Biotechnol. 21, 52-56 (2003).</li><li>Ogawa, S., Tomita, M., Shimizu, K., Yoshizato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon:+production+of+recombinant+human+serum+albumin.">Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin.</a> J Biotechnol. 128, 531-544 (2007).</li><li>Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+germline+transgenic+silkworm+that+secretes+recombinant+proteins+in+the+sericin+layer+of+cocoon.">A germline transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon.</a> Transgenic Res. 16, 449-465 (2007).</li><li>Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+alveolar+proteinosis+as+an+autoimmune+disease+with+neutralizing+antibody+against+granulocyte/macrophage+colony-stimulating+factor.">Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor.</a> J Exp Med. 190, 875-880 (1999).</li><li>Tanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lungs+of+patients+with+idiopathic+pulmonary+alveolar+proteinosis+express+a+factor+which+neutralizes+granulocyte-macrophage+colony+stimulating+factor.">Lungs of patients with idiopathic pulmonary alveolar proteinosis express a factor which neutralizes granulocyte-macrophage colony stimulating factor.</a> FEBS Lett. 442, 246-250 (1999).</li><li>Brown, C., Pihl, C., Murray, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+soluble+granulocyte-macrophage+colony-stimulating+factor+receptor:+identification+of+the+functional+ligand-binding+species.">Oligomerization of the soluble granulocyte-macrophage colony-stimulating factor receptor: identification of the functional ligand-binding species.</a> Cytokine. 9, 219-225 (1997).</li><li>Urano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-free+assay+to+estimate+the+neutralizing+capacity+of+granulocyte-macrophage+colony-stimulating+factor+autoantibodies.">A cell-free assay to estimate the neutralizing capacity of granulocyte-macrophage colony-stimulating factor autoantibodies.</a> J Immunol Methods. 360, 141-148 (2010).</li><li>Hayashida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+a+second+subunit+of+the+receptor+for+human+granulocyte-macrophage+colony-stimulating+factor+(GM-CSF):+reconstitution+of+a+high-affinity+GM-CSF+receptor.">Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor.</a> Proc Natl Acad Sci U S A. 87, 9655-9659 (1990).</li><li>Hansen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+GM-CSF+receptor+complex+reveals+a+distinct+mode+of+cytokine+receptor+activation.">The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation.</a> Cell. 134, 496-507 (2008).</li></ol>

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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