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

Siamo molto grati a K. Nakagaki, Dr. H. Ishii, il Dr. K. Suzuki, A. Yamagata, K. Oofusa per i loro preziosi contributi.

1. Produzione e purificazione di sGMRα <ol><li> Amplifica cDNA di sGMRα da placenta umana biblioteca di cDNA mediante reazione a catena della polimerasi (PCR). </li><li> Aggiungere 50 di base 5&#39;-UTR sequenza di fine baculovirus polyhedrin e 27 RGS base di codifica His-tag per terminare il 3 &#39;per l&#39;amplicone PCR da un altro PCR. </li><li> Inserire il prodotto amplificato di PCR in un pMSG1.1MG plasmide. </li><li> Iniettare il psGMR/M1.1MG costruire con il vettore helper pHA3PIG in uova di baco da seta PND...

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Il cell-free saggio stimato la capacità neutralizzante di GM-CSF autoanticorpi con eccellente riproducibilità e rapidità. L&#39;inibizione vincolante da GM-CSF autoanticorpi o frazioni siero del paziente IgG è stata valutata da questa analisi. I dati hanno mostrato una correlazione tra l&#39;inibizione legame della cellula senza test e l&#39;inibizione della crescita di un saggio biologico con cellule TF-1, rispettivamente. Il biologico è stato ampiamente utilizzato, ma nutrito difficoltà inerenti alla comparazion...

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

SFONDI: In precedenza, abbiamo dimostrato che la capacità di neutralizzare, ma non la concentrazione di GM-CSF autoanticorpi è stata correlata con la gravità della malattia nei pazienti con autoimmuni proteinosi alveolare polmonare (PAP) 1-3. Come abrogazione di GM-CSF bioattività nel polmone è la causa probabile per PAP autoimmuni 4,5, è promettente per misurare la capacità neutralizzante di GM-CSF autoanticorpi per valutare la gravità della malattia in ogni paziente con PAP. Fino ad oggi, la capacità neutralizzante di GM-CSF autoanticorpi è stata valutata mediante la valutazione della inibizione della crescita di cellule di midollo osseo umano o TF-1 cellule stimolate con GM-CSF 6-8. Nel sistema biologico, tuttavia, è spesso problematico ottenere dati affidabili e di confrontare i dati provenienti da diversi laboratori, a causa delle difficoltà tecniche nel mantenimento delle cellule in una condizione costante. OBIETTIVO: Per simulare GM-CSF legame con GM-CSF recettore sulla superficie cellulare tramite cell-free recettoriale-test. METODI: baco da seta transgenici tecnologia è stata applicata per ottenere una grande quantità di ricombinante solubile GM-CSF recettore alfa (sGMRα) con elevata purezza 9-13. Il sGMRα ricombinante era contenuta negli strati sericina idrofila di fili di seta, senza essere fuse alle proteine ​​della seta, e quindi, si può facilmente estrarre dai bozzoli in purezza buona con neutro soluzioni acquose 14,15. Fortunatamente, le strutture oligosaccaridi, che sono critici per il legame con GM-CSF, sono più simili alle strutture di sGMRα umane rispetto a quelle prodotte da altri insetti o lieviti. RISULTATI: La cella priva di sistema di analisi utilizzando sGMRα dato i dati con elevata plasticità e affidabilità. Vincolanti per sGMRα GM-CSF è stato dose-dipendente inibita da policlonali GM-CSF autoanticorpi in maniera simile al test biologico con cellule TF-1, indicando che il nostro nuovo sistema di test cell-free utilizzando sGMRα è più utile per la misurazione di attività neutralizzante di GM-CSF autoanticorpi rispetto al sistema biologico utilizzando TF-1 di cellule umane o cellule del midollo osseo. CONCLUSIONI: Abbiamo stabilito una cella priva di test quantificare la capacità neutralizzante di GM-CSF autoanticorpi.

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

Abbiamo progettato una cella priva di test legame recettore al fine di valutare il legame di granulociti-macrofagi colony-stimulating factor (GM-CSF) ai recettori. Ci permette di valutare inibizione competitiva del biotinilato GM-CSF legame al recettore alfa solubili GM-CSF da GM-CSF autoanticorpi con eccellente riproducibilità.

Un saggio gratuito Cell System Stima della capacità neutralizzante di GM-CSF utilizzando anticorpi ricombinanti solubili GM-CSF Receptor

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

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