We produce this MAb by culturing the hybridoma line in CL1000 flasks using DMEM (the specific formulation provided by ATCC) supplemented with 20% FBS & antibiotics. We purify the antibody by rProtein A affinity chromatography (eluted using 0.1 M sodium citrate buffer at pH 5.5). The eluted antibody is then dialyzed against PBS pH 7.4 and the sample becomes turbid. The ELISA titer of the pre dialyzed material is far better than that post dialysis. Any suggestions will be greatly appreciated.
Your elution method is quite mild and really cannot be any gentler, at least not when using protein A. Prolonged exposure to pH 5.5 however may cause precipitation. I would adjust the pH immediately after elution as it seems you are just relying on dialysis? Increasing or decreasing the salt concentration may help, depending on the mechanism of the interaction between the antibody molecules. Adding glycerol to 10% or detergent to 0.1% is worth a try too. These treatments have helped with various dialysis issues that I’ve seen over the years but may or may not work with this Ab. Finally, what is the concentration of the antibody? You may need to reduce the concentration, at least until you have found suitable dialysis conditions.
As you have 20% FBS in your culture, there should be tons of bovine IgG in your prep, and you thus might enrich bovine IgG.
Can you resort to immobilized anti-mouse instead? Or even immobilized antigen, if available?
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