Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.
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.
Hessing
M
M
Biotechnological Research Unit, Organon Teknika, Boxtel, Netherlands.
van Schijndel
H B
HB
van Grunsven
W M
WM
Wolf
H
H
Middeldorp
J M
JM
D000818:Animals; D000956:Antigens, Viral / Q000302:isolation & purification*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004854:Herpesvirus 4, Human / Q000276:immunology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000302:isolation & purification*; D014763:Viral Matrix Proteins
D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins