Dear Scott,
I try to answer short but in a way that it helps.
First, there are really good laboratory manuals available for the
production and handling of mAbs. E.G.: Harlow and Lane. "Antibodies, a
laboratory manual."
1) For direct labelling, the antibody should be as pure as possible,
otherwise you can get unefficient coupling and background if u use it.
2) Prior to purification you have to adopt the hybridomas to serum free
(SF) medium, as serum contains loads of Igs. This can take a while. I
think its still recommended to adopt them slowly i.e. mix serum
containig and serum free media successivly, the hybridomas must be given
time to adopt ( about a week for every mixture). There are special serum
free hybridoma media on the market.
Another, faster strategy which I never tried, is to grow large amounts
of cells, spin them wash them and resuspend them in SF medium and grow
them as long as they are producing antibody, then chuck them.
The antibodies are purified from the sup.
3) SF media usually contain just few protein (the one I used just
contained Insulin and Transferrin, Gibco) I purified the mAb then by
gelfiltraion (Superdex 200)
I have no experience with Cy3-labelling
Regards, Ina
--
Ina Hinners
ICRF
Secretory Pathways Laboratory
44 Lincolns Inn Fields
WC2A 3PX London
UK
email: I.Hinners at icrf.icnet.uk
