| Author/Editor | Smilović, Vanja | |
| Title | Razvoj celične linije za določanje biološke aktivnosti interferonov | |
| Translated title | Development of cell line suitable for the determination of biological activity of interferons | |
| Type | monografija | |
| Place | Ljubljana | |
| Publisher | Univerza v Ljubljani, Medicinska fakulteta | |
| Publication year | 2006 | |
| Volume | str. 106 | |
| Language | slo | |
| Abstract | Inteferons are cytokines playing an important role in immune response and defence against viruses. Some interferons represent a basis for important biopharmaceutics like Intron A for curing hepatitis C, and Avonex for curing multiple sclerosis. The European Pharmacopeia sugests that biological activity of interferons should be determined by AntiViral Assay (AVA). AVA includes the use of viruses classified as biosafety level 2, which significantly limits the wider use of this assay. To circumvent this obstacle, a reporter cell line, based on CHO-K1 cells (Chinese Hamster Ovary) was developed. This cell line is classified as biosafety level 1 and is therfore allowed to be used in a standard microbiological laboratory. The cell line was established by transferring pISRE-SEAP-Neo plasmid into CHO-K1 cells. Stable clones were selected and tested for IFN sensitivity. The most sensitive cell line was selected and designated as CHO-ISRE-SEAP test line. In this cell line, a reporter gene SEAP (Secretory Alkaline Phosphatase) is uder control of ISRE (Interferon-Stimulated Response Element), meaning that in certain concentration range the amount of SEAP is proportional to the quantity of IFN used for stimulation of cells. The detection limit for IFN-alpha is approximately 50 ng/mL and for IFN-beta 5 ng/mL. It is suprising that the sensitivity of the new reporter cell line for IFN-beta is ten times higher as compared to sensitivity for IFN-alpha. The new reporter gene assay (RGA) for IFN-alpha or IFN-beta, based on CHO-ISRE-SEAP test line, appears to be rapid, less expensive and more practical as compared to AVA. We therefore find it very appropriate for routine determination of biological activity in vitro for a variety of samples containing IFN-alpha or IFN-beta. However, there is a limitation in use, namely in samples containing both cytokines, neither RGA nor AVA can differentiate them. | |
| Descriptors | CELL LINE INTERFERON-ALPHA INTERFERON TYPE II CHO CELLS PLASMIDS TRANSFECTION CULTURE MEDIA POLYMERASE CHAIN REACTION |