Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells

URN to cite this document:

urn:nbn:de:bvb:355-epub-204249 Copy

DOI to cite this document:

10.5283/epub.20424 Copy

Hessing, M. ; van Schijndel, H. B. ; van Grunsven, W. M. ; Wolf, Hans J. ; Middeldorp, J. M.

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Date of publication of this fulltext: 11 Apr 2011 10:53

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Details

Item type:	Article
Journal or Publication Title:	Journal of chromatography
Publisher:	Elsevier
Volume:	599
Number of Issue or Book Chapter:	1-2
Page Range:	pp. 267-272
Date:	1992
Institutions:	Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene
Identification Number:	Value Type 1320046 PubMed ID
Classification:	Notation Type Animals MESH Antigens, Viral/isolation & purification MESH Blotting, Western MESH CHO Cells MESH Cricetinae MESH Electrophoresis, Polyacrylamide Gel MESH Enzyme-Linked Immunosorbent Assay MESH Herpesvirus 4, Human/immunology MESH Recombinant Proteins/isolation & purification MESH Viral Envelope Proteins/isolation & purification MESH Viral Matrix Proteins MESH
Dewey Decimal Classification:	600 Technology > 610 Medical sciences Medicine
Status:	Published
Refereed:	Unknown
Created at the University of Regensburg:	Unknown
Item ID:	20424

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Abstract

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 ...

Abstract

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.

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Metadata last modified: 26 Nov 2020 07:06

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