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从亚急性硬化性全脑炎(SSPE)和多发性硬化症(MS)患者大脑中提取和纯化活性免疫球蛋白G(IgG)

Extraction and purification of active IgG from SSPE and MS brain.

作者信息

Owens G P, Burgoon M P, Devlin M E, Gilden D H

机构信息

Department of Neurology, University of Colorado Health Sciences Center, Denver 80262, USA.

出版信息

J Virol Methods. 1997 Nov;68(2):119-25. doi: 10.1016/s0166-0934(97)00118-3.

Abstract

Immunoglobulin (Ig) G was purified from soluble and membrane fractions of postmortem subacute sclerosing panencephalitis (SSPE) brain, multiple sclerosis (MS) brain plaque-periplaque white matter, and normal human brain (NHB) white matter. After homogenization in 0.32 M sucrose and removal of cell debris and nuclei by low-speed centrifugation, soluble and crude membrane fractions were separated by ultracentrifugation. After removal of sucrose by dialysis, IgG was isolated from the soluble fraction by protein A affinity chromatography. IgG was obtained from the membrane fraction by elution at low pH and purification from the eluate by protein A chromatography. Whereas very little IgG was in NHB white matter, significant levels of IgG were recovered from both SSPE and MS brain. Both immunocytochemical staining of measles virus-infected cells in tissue culture and protein immunoblotting of virus-infected cell lysates showed that the IgG from SSPE brain contained activity specific for measles virus protein. The abundance, purity and functional activity of IgG extracted from SSPE and MS brain indicate that IgG extracted from the brain of humans with an inflammatory disease of unknown etiology can be used to identify its corresponding antigen.

摘要

免疫球蛋白(Ig)G从亚急性硬化性全脑炎(SSPE)脑、多发性硬化症(MS)脑斑块周围白质以及正常人类脑(NHB)白质的可溶性和膜组分中纯化得到。在0.32M蔗糖中匀浆并通过低速离心去除细胞碎片和细胞核后,通过超速离心分离可溶性和粗膜组分。通过透析去除蔗糖后,通过蛋白A亲和色谱从可溶性组分中分离IgG。通过低pH洗脱从膜组分中获得IgG,并通过蛋白A色谱从洗脱液中纯化。NHB白质中IgG含量极少,而从SSPE和MS脑中均回收了显著水平的IgG。组织培养中麻疹病毒感染细胞的免疫细胞化学染色以及病毒感染细胞裂解物的蛋白质免疫印迹均表明,来自SSPE脑的IgG含有针对麻疹病毒蛋白的特异性活性。从SSPE和MS脑中提取的IgG的丰度、纯度和功能活性表明,从未知病因的炎症性疾病患者脑中提取的IgG可用于鉴定其相应抗原。

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