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.
| Original language | English |
|---|---|
| Pages (from-to) | 267-72 |
| Number of pages | 6 |
| Journal | Journal of Chromatography B |
| Volume | 599 |
| Issue number | 1-2 |
| Publication status | Published - 22 May 1992 |
Keywords
- Animals
- Antigens, Viral
- Blotting, Western
- CHO Cells
- Cricetinae
- Electrophoresis, Polyacrylamide Gel
- Enzyme-Linked Immunosorbent Assay
- Herpesvirus 4, Human
- Journal Article
- Recombinant Proteins
- Viral Envelope Proteins
- Viral Matrix Proteins