The role of scavenger receptor BI in hepatitis - eTheses Repository ...
The role of scavenger receptor BI in hepatitis - eTheses Repository ...
The role of scavenger receptor BI in hepatitis - eTheses Repository ...
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Preparation <strong>of</strong> rat anti-E2 mAbs.<br />
65<br />
Anti-E2 and control mAbs were cloned from rats immunised with recomb<strong>in</strong>ant<br />
antigen as previously described (99, 130, 282). Hybridoma cells express<strong>in</strong>g<br />
the panel <strong>of</strong> antibodies were grown <strong>in</strong> m<strong>in</strong>iPERM bioreactors (Gre<strong>in</strong>er Bio<br />
One, Germany), accord<strong>in</strong>g to the manufacturers <strong>in</strong>structions, under the<br />
supervision <strong>of</strong> Dr. Margaret Goodall. Approximately 250ml <strong>of</strong> cell culture<br />
medium conta<strong>in</strong><strong>in</strong>g mAb was harvested from each bioreactor allow<strong>in</strong>g IgG<br />
isolation us<strong>in</strong>g the follow<strong>in</strong>g protocol.<br />
1. A 2ml column conta<strong>in</strong><strong>in</strong>g 750µl <strong>of</strong> Fast-flow prote<strong>in</strong> G conjugated<br />
sepharose beads (GE Healthcare, UK) was prepared for each mAb to<br />
be isolated.<br />
2. To capture IgG, the column was connected to a peristaltic pump<br />
(Pharmacia, Sweden) and 10ml phosphate buffered sal<strong>in</strong>e (PBS)<br />
(Gibco) passed through to wash, followed by 10ml <strong>of</strong> harvested culture<br />
media.<br />
3. <strong>The</strong> column was washed once more and the IgG eluted with 10ml 0.1M<br />
glyc<strong>in</strong>e (Sigma-Aldrich, MO, USA) at pH 2.7, the acidic eluate was<br />
immediately neutralised with 650µl 1M TRIS (Sigma-Aldrich) at pH 9.0.<br />
4. F<strong>in</strong>ally, the eluate was dialysed aga<strong>in</strong>st PBS overnight at 4ºC. IgG<br />
concentration was determ<strong>in</strong>ed us<strong>in</strong>g a UV spectrophotometer<br />
(Amersham, UK).