एक फ्री सेल परख जीएम-CSF एंटीबॉडी के उदासीन क्षमता का आकलन प्रणाली पुनः संयोजक घुलनशील रिसेप्टर जीएम-CSF का उपयोग
Summary
हम एक रिसेप्टर सेल मुक्त बाध्यकारी परख डिजाइन क्रम में रिसेप्टर्स करने के लिए granulocyte-बृहतभक्षककोशिका कॉलोनी उत्तेजक कारक (जीएम-CSF) के बंधन का अनुमान है. यह हमें सक्षम बनाता है biotinylated जीएम-CSF के प्रतिस्पर्धी निषेध घुलनशील जीएम-CSF रिसेप्टर अल्फा के लिए बाध्य उत्कृष्ट reproducibility के साथ जीएम-CSF autoantibody द्वारा मूल्यांकन है.
Abstract
BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.
Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.
OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.
METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMRα) with high purity9-13. The recombinant sGMRα was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMRα than those produced by other insects or yeasts.
RESULTS: The cell-free assay system using sGMRα yielded the data with high plasticity and reliability. GM-CSF binding to sGMRα was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMRα is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.
CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.
Protocol
Discussion
सेल मुक्त परख उत्कृष्ट reproducibility और तेज़ी के साथ जीएम-CSF autoantibodies के को निष्क्रिय क्षमता का अनुमान है. जीएम-CSF autoantibodies या रोगी के सीरम आईजीजी भिन्न बंधन निषेध इस परख द्वारा मूल्यांकन किया गया था. डेटा सेल मुक्त परख ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम उनके बहुमूल्य योगदान के लिए लालकृष्ण Nakagaki, डा. एच. इशी, डा. के सुजुकी, ए यामागाटा, लालकृष्ण Oofusa बहुत आभारी हैं.
Materials
| Name of reagent | Company | Catalog # | Comments |
| human placenta cDNA library | Takara | ||
| Nickel affinity column | GE Healthcare | 17-5247-01 | |
| biotin hydrazide (EZ-Link Biotin Hydrazide) | PIERCE | 21339 | |
| rhGM-CSF (leukine) | Genzyme Corporation | ||
| Nunc Immobilizer Amino | Nalge Nunc International | 436007 | |
| Monoclonal Anti-polyHistidine antibody produced in mouse | Sigma-Aldrich | H1029 | 0.2ml |
| blocking solution (StabilCoat) | Surmodics | SC01-1000 | 1000ml |
| ZyMAX Streptavidin-AP Conjugate | Invitrogen | 43-8322 | |
| CDP-Star Ready-to-Use With Sapphire-II | Applied Biosystems | T2214 | |
| chemiluminescence plate reader | BERTHOLD TECHNOLOGIES | TriStar LB 941 |
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