Hessing, M. and van Schijndel, H. B. and van Grunsven, W. M. and Wolf, Hans J. and Middeldorp, J. M.
Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.
Journal of chromatography 599 (1-2), pp. 267-272.
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.
|Institutions:|| Medicine > Lehrstuhl für Medizinische Mikrobiologie und Hygiene|
|Antigens, Viral/isolation & purification||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|
|Subjects:||600 Technology > 610 Medical sciences Medicine|
|Created at the University of Regensburg:||Unknown|
|Deposited On:||11 Apr 2011 10:53|
|Last Modified:||21 Jul 2011 02:10|