If monoclonal antibody would be obtained from ascites fluid during hybridoma expression in vivo -> protein A/G-sepharose purification, how much non-specific AB contamination and non-target proteins presence in the product could be? Will target AB mass percentage be =/>98%
Hi Leonid,
The monoclonal antibody prepared from acitic fluid via protein A/G carrier purification, can contain some antibodies, which are non-specific to your antigen. They may present in the ascitic fluid and will also bind to the protein A/G carrier. The class of these antibodies will depend on the kind of the carrier ligand (A or G). Besides that, the antibody preparations can contain some proteins that specifically bind to their Fc parts (via non-protein A/G binding sites), if you don't wash the column with adsorbed mAbs thoroughly (not only with a loading buffer, but also with NaCl solution) before eluting your mAbs. And, an extremely rare case, if your mAb has a specifity not only to your target protein, but also to a protein that may exist in the ascitic fluid (as you know, all antibodies may have a certain variety of specificities due to the ability of binding antigens by their different CDRs).
Thanks! And if AB would be expressed in vitro culture of hybridoma such disadvantages will not appear or purification on antigen-sepharose still needed?
Such disadvantages may not appear in mAbs prepared from in vitro hybridoma cultures, but to my mind, I would check it anyway, if highly purified preparations are needed. For hybridoma-prepared mAbs, removal of culture medium proteins is needed. But if these proteins don't interfere with Ab-Ag binding, presense of certain amounts of them does not matter. If about 90% mAb purity is OK (with no interfering components), I would not perform an additional mAb purification on an antigen carrier, it is a rather harsh procedure, some Abs may not survive.
- Badges
- Science topic