Do you think that it is safe to use human mAbs from EBV transformed memory B cells as tools for flow cytometry? What is the probability of EBV contamination in these preparations? Are there any procedures to get rid of the virus?
Thank you very much for your feedback,
I assume that you are concerned mostly about contaminating your assays with EBV antigens and creating false positives? Or are you concerned about creating contagious assay materials that could infect lab staff?
Most of those clones will have integrated EBV genome(s).
Article When Epstein-Barr virus persistently infects B-cell lines, i...
The cell lines should be expected to produce EBV antigens, and can be contagious to humans. Many proteins should be expected in preparations. Article Proteins of purified Epstein–Barr virus
mAbs vary a lot in purification level. The primary technique is chromatography.
Article Separation techniques: Chromatography
This does a very good job, and you shouldn't get virus in those preps. But you may get proteins of similar size and gross hydrophobicity etc..You can test your preparations with a Nanodrop or other spectrometer to see how pure your mAbs are. You will need to know what animal cell, what mAb type (generally IgG except for specialty) and subtype (IgG1, 2, 3, or 4). From that you can calculate (or look up) the size of the molecule, and what it's peak and spectral characteristics should be. I like the Nanodrop because it takes so little material, and it's fast. (But it's possible to screw it up or mistreat the equipment so it gets dried junk on it, and bad results.)
You can also test your mAb preps with EBV antibodies to see if that turns up anything.
The other purification techniques are antigen binding columns, and heavy chain affinity columns. These can be considered to fall under chromatography. The former is done for the highest quality antivenins for instance. They create columns using snake venom and after binding the antibodies, wash and elute. The same thing can be done for marker mAbs, but rarely is. Protein G, Bip, Trim21, streptavidin, etc. can be used to bind IgG to columns, wash and elute.
Hi Brian, many thanks for your valuable comments.
I'm mostly concerned with three practical issues:
1. how do the titers of mAbs produced by EBV transformed cells relate to those produced by hybridoma?
2. are these EBV transformed cells stable producers in long term culture, can one culture them in capillary systems?
3. can one efficiently get rid of EBV particles from these mAb preps so that they can safely be used in downstream applications?
1. I'm not sure, and it's probably pretty variable. I found this paper. Article Regulation of antibody production by an antigen-specific EBV...
2. I would think so, as long as they don't get a tolerizing signal to stop production.
3. This was answered above.
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