PURIFICATION AND QUANTIFICATION OF RECOMBINANT EPSTEIN-BARR VIRAL GLYCOPROTEINS GP350/220 FROM CHINESE-HAMSTER OVARY CELLS

被引：10

作者：

HESSING, M ^{[1
]}

VANSCHIJNDEL, HB ^{[1
]}

VANGRUNSVEN, WMJ ^{[1
]}

WOLF, H ^{[1
]}

MIDDELDORP, JM ^{[1
]}

机构：

[1] MAX VON PETTENKOFER INST,MUNICH,GERMANY

来源：

JOURNAL OF CHROMATOGRAPHY | 1992年 / 599卷 / 1-2期

关键词：

D O I：

10.1016/0021-9673(92)85479-D

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline. concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.

引用

页码：267 / 272

页数：6

共 35 条

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