I am aware of Streptavidin magnetic beads which can be used with biotinylated antibody in MACS. But I would like to know if anyone has tried isolating or depleting cells from heterogeneous population by labeling cells with purified antibody followed by use of protein A magnetic beads.
Neeraj - this is an interesting question. I have a couple of thoughts, but first would ask a question.
Why can't you use a streptavidin/biotin system? I've seen this come up in samples (especially food samples) where there is a large amount of pre-existing biotin that competes for the the magnetic beads. Is that the reason, or is there some other motivation for using a non-streptavidin system?
That said, I think it might be worth a try, but I'd raise a concern to watch out for. At least as I envision it, the streptavidin/biotin system is a bit 'forgiving' in that commercially available SA is often tetravalent (that is, it can bind up to 4 biotin molecules) and the biotinylation protocols used for most antibody conjugation produces 3-7 biotins per Ab molecule. As a result, there are a lot of orientations of the streptavidin and biotinylated antibody that will permit binding. With protein A, because there is only one steric 'handshake' between the protein A molecule and the Fc portion of the IgG molecule, the opportunity for binding would appear to be significantly less. Remember, unlike protein A display on a bacterial cell wall, on a magnetic bead surface the protein A will be randomly oriented and as such will not always display its Fc-engaging face.
If streptavidin-biotin is not possible, and you don't have acceptable yield with your protein A strategy, you might have success with a particle on which the antibody has been directly linked. My company does not provide magnetic particles with this functionality, but can produce custom buoyant microbubbles that might suit your experimental needs.
Good luck, and let me know if I can help,
John Younger
www.akadeum.com
Fantastic John. BACS was something i did not really know about. First I will answer your query. I am aware of Streptavidin-biotin system and am using it, but cannot go beyond a round of positive selection. Say I want to isolate double positive cells for two antigens. FACS is an option, but I want to bypass FACS because of technicalities with using the machine, amount of time and labour invested, and at times the availability of sorter.
BACS is very interesting and I feel now I will be able to go upto triple positive. So the idea is to perform one round of positive selection with streptavidin biotin and then use Protein A beads for second round of positive selection.
I think it will be interesting if you can let me know if you can directly conjugate antibody to your microbubbles.
Best,
Neeraj
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