一个细胞检测系统估算的GM - CSF抗体的中和能力，采用重组可溶性GM - CSF受体
Summary
我们设计了一个无细胞受体结合实验，以估计约束力的粒细胞 – 巨噬细胞集落刺激因子（GM – CSF的）受体。它使我们能够评估竞争性抑制GM – CSF的抗体生物素标记的GM – CSF的可溶性GM – CSF受体α的结合，具有优良的重现性。
Abstract
背景：在此之前，我们表明，中和能力，但没有GM – CSF的抗体浓度是在患者自身免疫性肺泡蛋白沉积症（PAP）的1-3疾病的严重程度相关。废止的GM – CSF在肺癌的生物活性可能导致自身免疫性PAP 的 4,5，它是有前途的GM – CSF自身抗体的中和能力的评估与人民行动党在每个病人的病情轻重程度来衡量。
到现在为止，已评定的GM – CSF自身抗体的中和能力评估人类骨髓细胞或TF – 1 细胞 6-8与GM – CSF的刺激的生长抑制。然而，在生物测定系统，它往往是问题，以获得可靠的数据，以及比较来自不同实验室的数据，由于技术上的困难，保持在一个恒定的条件下的细胞。
目的：为了模仿GM – CSF，GM – CSF受体细胞表面上的具有约束力的使用无细胞受体结合测定。
方法：转基因家蚕技术是获得高纯度9-13的重组可溶性GM – CSF受体α（sGMRα）为大量的应用。重组sGMRα载而不融合丝蛋白在亲水丝胶丝线层，因此，我们可以很容易地从良好的纯度蚕茧14,15与中性水溶液中提取。幸运的是，寡糖的结构，这是与GM – CSF结合的关键，更类似于人类sGMRα的结构比其他昆虫或酵母生产。
结果：无细胞检测系统使用sGMRα取得的数据，具有很高的可塑性和可靠性。 GM – CSF的剂量依赖性绑定sGMRα以类似的方式使用TF – 1细胞的生物活性，抑制多克隆GM – CSF的抗体，这表明我们的新的无细胞检测系统使用sGMRα是中和活性的测量更为有用GM – CSF的自身抗体比使用TF – 1细胞或骨髓细胞的生物测定系统。
结论：我们建立了无细胞的检测，量化的GM – CSF抗体的中和能力。
Protocol
1。生产和净化的sGMRα 从人胎盘cDNA文库中扩增cDNA的聚合酶链反应（PCR）的sGMRα。 50基地5' – UTR序列杆状病毒多角月底和27基编码的RGS His – tag的3'末端的另一个PCR扩增PCR扩增。 插入质粒pMSG1.1MG的扩增的PCR产物。 注入鸡蛋蚕PND – W1株pHA3PIG助手载体的建设psGMR/M1.1MG。 后部孵化G0期幼虫蛾25 ° C。 G1的屏幕获得通过交配，兄弟姐妹之间或与PND – W1在眼里MGFP表达，获…
Discussion
无细胞检测估计具有优良的重现性和快速性的GM – CSF的自身抗体的中和能力。结合抑制GM – CSF的自身抗体或患者的血清IgG分数是评估这个实验。数据显示，结合抑制细胞免费检测和使用TF – 1细胞，分别为生物活性的生长抑制之间的关系。已被广泛使用的生物活性，但窝藏在比较数据之间不同的设施和不同的时间点，我们通过使用这个新的系统可避免的困难。
GM – CSF的结合sGMRα?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们非常感谢他们的宝贵贡献，K. Nakagaki，博士阁下石井，铃木博士光，A.山形，光Oofusa。
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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Tags
Cell-free Assay SystemNeutralizing CapacityGM-CSF AntibodyRecombinant Soluble GM-CSF ReceptorAutoimmune Pulmonary Alveolar Proteinosis (PAP)Disease SeverityGM-CSF BioactivityNeutralizing Capacity AssessmentGrowth InhibitionHuman Bone Marrow CellsTF-1 CellsBioassay SystemCell-free Receptor-binding AssayTransgenic Silkworm TechnologyRecombinant SGMRαSilk ThreadsCocoons
Cite This Article
Urano, S., Tazawa, R., Nei, T., Motoi, N., Watanabe, M., Igarashi, T., Tomita, M., Nakata, K. A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor. J. Vis. Exp. (52), e2742, doi:10.3791/2742 (2011).